Virus vectors for targeting ophthalmic tissues

ABSTRACT

The present disclosure provides AAV capsid proteins comprising a modification in the amino acid sequence and virus vectors comprising the modified AAV capsid protein. The disclosure also provides methods of administering the vims vectors and vims capsids of the disclosure to a cell or to a subject in vivo.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Ser.No. 62/652,108, filed Apr. 3, 2018, which is incorporated by referenceherein in its entirety for all purposes.

FIELD OF THE DISCLOSURE

The present disclosure relates to modified capsid proteins fromadeno-associated virus (AAV) and virus capsids and virus vectorscomprising the same. In particular, the disclosure relates to modifiedAAV capsid proteins and capsids comprising the same that can beincorporated into viral vectors to confer a desirable transductionprofile with respect to a target tissue(s) of interest, such as anophthalmic tissue.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 3, 2019, isnamed STRD-007_01 WO_ST25.txt and is 652 kb in size.

BACKGROUND

New adeno-associated virus (AAV) strains isolated from animal tissuesand adenoviral stocks have expanded the panel of AAV vectors availablefor therapeutic gene transfer applications. Comprehensive efforts to maptissue tropisms of these AAV isolates in animal models are currentlyunderway. The ability to direct homing of AAV vectors to selectiveorgans is useful for gene therapy and other therapeutic applications.

Gene therapy shows promise for treating ophthalmic diseases, and it hasbeen shown that AAV is capable of transducing multiple cell types andcell layers in the eye. The compositions and methods provided hereinaddress a need in the art for nucleic acid delivery vectors withdesirable targeting features, such as targeting to specific regions ofthe eye (e.g., the retinal pigment epithelium (RPE) or retina).

BRIEF SUMMARY

Provided herein are AAV capsid proteins and AAV viral vectors thatspecifically target and infect one or more tissues of interest, such astissues of the eye (e.g., the RPE or retina). In some embodiments, thepresent disclosure provides a recombinant adeno-associated virus (AAV)capsid protein comprising one or more amino acid substitutions, whereinthe one or more amino acid substitutions modify one or moresurface-exposed loops on the AAV capsid protein. In some embodiments,the modification of the one or more surface-exposed loops results in anenhanced transduction profile with respect to a target tissue. Inembodiments, when the AAV capsid protein is incorporated into an AAVvector, the modification of the one or more surface-exposed loopsresults in enhanced transduction to an ophthalmic tissue. In someembodiments, when the AAV capsid protein incorporated into an AAVvector, the modification of the one or more surface-exposed loopsresults in enhanced transduction to the retinal pigment epithelium (RPE)and/or the retina as compared to a parental capsid which does notcomprise the modification.

In some embodiments, the present disclosure provides a recombinant AAVcapsid protein, wherein the capsid protein comprises a substitution in asurface-exposed loop of the AAV capsid protein, wherein the substitutionhas a sequence of any one of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21,22, 187, 188, 189, 190, 191, 192, 193, and 194. In some embodiments, therecombinant AAV capsid comprises an amino acid sequence with at least80% sequence identity to a capsid protein of any one of AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8,AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV and avian AAV. In someembodiments, the recombinant AAV capsid protein comprises an amino acidsequence that has at least 90% sequence identity, with any one of SEQ IDNO: 11-12, 23-49, or 195-254.

In some embodiments, the disclosure provides an AAV capsid proteincomprising an amino acid substitution, wherein the amino acidsubstitution replaces the amino acids in the region corresponding toamino acid positions 591-596 of SEQ ID NO. 11 or amino acids 590-595 ofSEQ ID NO. 12.

In some embodiments, the disclosure provides a recombinant AAV capsidprotein, wherein the capsid protein comprises a substitution comprisinga sequence of six amino acids (X¹-X²-X³-X⁴-X⁵-X⁶) that does not occur inthe native AAV capsid protein, wherein X² is V and X⁵ is L (SEQ IDNO:186). In some embodiments, the substitution is in a surface-exposedloop of the AAV capsid protein.

In some embodiments, the disclosure provides an AAV capsid protein,wherein the capsid protein comprises the amino acid sequence of any oneof SEQ ID NO: 11-12, 23-49, or 195-254. In some embodiments thedisclosure provides an AAV viral vector comprising an AAV capsid proteinthat comprises an amino acid sequence at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto the sequence of any one of SEQ ID NO: 11-12, 23-49, or 195-254. Insome embodiments, the AAV viral vector specifically targets and infectsthe RPE and/or the retina. In some embodiments, the AAV viral vectordemonstrates increased targeting and infection of the RPE and/or theretina as compared to a parental or wildtype AAV viral vector.

The present disclosure also provides a nucleotide sequence, or anexpression vector comprising the same, that encodes one or more of therecombinant AAV capsid proteins of the disclosure. The presentdisclosure also provides a cell that comprises one or more nucleotidesequences or expression vectors of the disclosure.

The present disclosure also provides an AAV capsid comprising arecombinant AAV capsid protein of this disclosure. Further provided is aviral vector comprising a recombinant AAV capsid of this disclosure aswell as a composition comprising a recombinant AAV capsid protein, AAVcapsid and/or viral vector of this disclosure in a pharmaceuticallyacceptable carrier.

The present disclosure additionally provides a method of introducing anucleic acid into a cell, comprising contacting the cell with the viralvector of this disclosure. The cell can be in a subject and in someembodiments, the subject can be a human subject.

In some embodiments, a method of treating a patient in need thereof isprovided, the method comprising administering to the patient atherapeutically effective amount of an AAV viral vector of thedisclosure.

These and other aspects are addressed in more detail in the detaileddescription set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B. Bubble plots showing analysis of library diversity,directed evolution and enrichment of novel antigenic footprints.Parental (FIG. 1A) and evolved (FIG. 1B) libraries were subjected tohigh-throughput sequencing using the Illumina MiSeq platform. Followinganalysis with a custom Perl script, enriched amino acid sequences wereplotted. Each bubble represents a distinct capsid amino acid sequencewith the radius of the bubble proportional to the number of reads forthat variant in the respective library. The y-axis represents theabsolute number of reads, transformed to log base 2. Data are spreadalong the x-axis for ease of visualization. The percent reduction inunique clones (70.3%) directly demonstrates that numerous “un-fit”sequences were removed after a first round of evolution.

FIG. 2A and FIG. 2B. Bubble plots showing parental (FIG. 2A) and evolved(FIG. 2B) libraries for a first round of evolution. FIG. 2A and FIG. 2Bshow the same data as in FIG. 1A and FIG. 1B, respectively, but the datahas been normalized to percent total reads, allowing for longitudinalcomparison across subsequent rounds of evolution.

FIG. 3A and FIG. 3B. Bubble plots showing parental (FIG. 3A) and evolved(FIG. 3B) libraries for a third round of evolution performed in theretina, normalized to percent total reads.

FIG. 4A and FIG. 4B. Bubble plots showing parental (FIG. 3A) and evolved(FIG. 3B) libraries for a third round of evolution performed in the RPE,normalized to percent total reads.

FIG. 5. Graph showing transduction of various AAV-luciferase vectorscomprising mutant capsid proteins (STRD-1, SEQ ID NO: 34; STRD-2, SEQ IDNO: 32; STRD-3, SEQ ID NO: 36; STRD-4, SEQ ID NO: 195; STRD-5, SEQ IDNO: 35; STRD-6, SEQ ID NO: 196; STRD-7, SEQ ID NO: 37; and STRD-8, SEQID NO: 40) into U87 cells in culture. The cells were infected at amultiplicity of infection (MOI) of 10,000 vg/cell. Relative light units(RLUs) were measured 48 hours post-transduction.

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. The terminology used in thedetailed description herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

All publications, patent applications, patents, GenBank or otheraccession numbers and other references mentioned herein are incorporatedby reference herein in their entirety.

The designation of all amino acid positions in the AAV capsid proteinsin the disclosure and the appended claims is with respect to VP1 capsidsubunit numbering. It will be understood by those skilled in the artthat the modifications described herein if inserted into the AAV capgene may result in modifications in the VP1, VP2 and/or VP3 capsidsubunits. Alternatively, the capsid subunits can be expressedindependently to achieve modification in only one or two of the capsidsubunits (VP1, VP2, VP3, VP1+VP2, VP1+VP3, or VP2+VP3).

Definitions

The following terms are used in the description herein and the appendedclaims:

The singular forms “a,” “an” and “the” are intended to include theplural forms as well, unless the context clearly indicates otherwise.

Furthermore, the term “about” as used herein when referring to ameasurable value such as an amount of the length of a polynucleotide orpolypeptide sequence, dose, time, temperature, and the like, is meant toencompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% ofthe specified amount.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Unless the context indicates otherwise, it is specifically intended thatthe various features described herein can be used in any combination.

Moreover, the present disclosure also contemplates that in someembodiments, any feature or combination of features set forth herein canbe excluded or omitted. To illustrate further, if, for example, thespecification indicates that a particular amino acid can be selectedfrom A, G, I, L and/or V, this language also indicates that the aminoacid can be selected from any subset of these amino acid(s) for exampleA, G, I or L; A, G, I or V; A or G; only L; etc., as if each suchsubcombination is expressly set forth herein. Moreover, such languagealso indicates that one or more of the specified amino acids can bedisclaimed. For example, in particular embodiments the amino acid is notA, G or I; is not A; is not G or V; etc., as if each such possibledisclaimer is expressly set forth herein.

As used herein, the terms “reduce,” “reduces,” “reduction” and similarterms mean a decrease of at least about 10%, about 15%, about 20%, about25%, about 35%, about 50%, about 75%, about 80%, about 85%, about 90%,about 95%, about 97% or more.

As used herein, the terms “increase,” “improve,” “enhance,” “enhances,”“enhancement” and similar terms indicate an increase of at least about10%, about 15%, about 20%, about 25%, about 50%, about 75%, about 100%,about 150%, about 200%, about 300%, about 400%, about 500% or more.

The term “parvovirus” as used herein encompasses the familyParvoviridae, including autonomously replicating parvoviruses anddependoviruses. The autonomous parvoviruses include members of thegenera Protoparvovirus, Erythroparvovirus, Bocaparvovirus, andDensovirus subfamily. Exemplary autonomous parvoviruses include, but arenot limited to, minute virus of mouse, bovine parvovirus, canineparvovirus, chicken parvovirus, feline panleukopenia virus, felineparvovirus, goose parvovirus, H1 parvovirus, muscovy duck parvovirus,B19 virus, and any other autonomous parvovirus now known or laterdiscovered. Other autonomous parvoviruses are known to those skilled inthe art. See, e.g., BERNARD N. FIELDS et al, VIROLOGY, volume 2, chapter69 (4th ed., Lippincott-Raven Publishers; Cotmore et al. Archives ofVirology DOI 10.1007/s00705-013-1914-1).

As used herein, the term “adeno-associated virus” (AAV), includes but isnot limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3Aand 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAVtype 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV typerh32.33, AAV type rh8, AAV type rh10, AAV type rh74, AAV type hu.68,avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV,bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAVPHP. B, and any other AAV now known or later discovered. See, e.g.,BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed.,Lippincott-Raven Publishers). A number of AAV serotypes and clades havebeen identified (see, e.g., Gao et al, (2004) J. Virology 78:6381-6388;Moris et al, (2004) Virology 33-:375-383; and Table 2).

As used herein, the term “chimeric AAV” refers to an AAV comprising acapsid protein with regions, domains, individual amino acids that arederived from two or more different serotypes of AAV. In someembodiments, a chimeric AAV comprises a capsid protein comprised of afirst region that is derived from a first AAV serotype and a secondregion that is derived from a second AAV serotype. In some embodiments,a chimeric AAV comprises a capsid protein comprised of a first regionthat is derived from a first AAV serotype, a second region that isderived from a second AAV serotype, and a third region that is derivedfrom a third AAV serotype. In some embodiments, the chimeric AAV maycomprise regions, domains, individual amino acids derived from two ormore of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10,AAV11, and/or AAV12. For example, the chimeric AAV may include regions,domains, and/or individual amino acids from a first and a second AAVserotype as shown below (Table 1), wherein AAVX+Y indicates a chimericAAV including sequences derived from AAVX and AAVY.

TABLE 1 Chimeric AAVs Second AAV Serotype AAV1 AAV2 AAV3 AAV4 AAV5 AAV6AAV7 First AAV Sertoype AAV1 x AAV1 + 2 AAV1 + 3 AAV1 + 4 AAV1 + 5AAV1 + 6 AAV1 + 7 AAV2 AAV2 + 1 x AAV2 + 3 AAV2 + 4 AAV2 + 5 AAV2 + 6AAV2 + 7 AAV3 AAV3 + 1 AAV3 + 2 x AAV3 + 4 AAV3 + 5 AAV3 + 6 AAV3 + 7AAV4 AAV4 + 1 AAV4 + 2 AAV4 + 3 x AAV4 + 5 AAV4 + 6 AAV4 + 7 AAV5 AAV5 +1 AAV5 + 2 AAV5 + 3 AAV5 + 4 x AAV5 + 6 AAV5 + 7 AAV6 AAV6 + 1 AAV6 + 2AAV6 + 3 AAV6 + 4 AAV6 + 5 x AAV6 + 7 AAV7 AAV7 + 1 AAV7 + 2 AAV7 + 3AAV7 + 4 AAV7 + 5 AAV7 + 6 x AAV8 AAV8 + 1 AAV8 + 2 AAV8 + 3 AAV8 + 4AAV8 + 5 AAV8 + 6 AAV8 + 7 AAV9 AAV9 + 1 AAV9 + 2 AAV9 + 3 AAV9 + 4AAV9 + 5 AAV9 + 6 AAV9 + 7 AAV10 AAV10 + 1 AAV10 + 2 AAV10 + 3 AAV10 + 4AAV10 + 5 AAV10 + 6 AAV10 + 7 AAV11 AAV11 + 1 AAV11 + 2 AAV11 + 3AAV11 + 4 AAV11 + 5 AAV11 + 6 AAV11 + 7 AAV12 AAV12 + 1 AAV12 + 2AAV12 + 3 AAV12 + 4 AAV12 + 5 AAV12 + 6 AAV12 + 7 Second AAV SerotypeAAV8 AAV9 AAV10 AAV11 AAV12 First AAV Sertoype AAV1 AAV1 + 8 AAV1 + 9AAV1 + 10 AAV1 + 11 AAV1 + 12 AAV2 AAV2 + 8 AAV2 + 9 AAV2 + 10 AAV2 + 11AAV2 + 12 AAV3 AAV3 + 8 AAV3 + 9 AAV3 + 10 AAV3 + 11 AAV3 + 12 AAV4AAV4 + 8 AAV4 + 9 AAV4 + 10 AAV4 + 11 AAV4 + 12 AAV5 AAV5 + 8 AAV5 + 9AAV5 + 10 AAV5 + 11 AAV5 + 12 AAV6 AAV6 + 8 AAV6 + 9 AAV6 + 10 AAV6 + 11AAV6 + 12 AAV7 AAV7 + 8 AAV7 + 9 AAV7 + 10 AAV7 + 11 AAV7 + 12 AAV8 xAAV8 + 9 AAV8 + 10 AAV8 + 11 AAV8 + 12 AAV9 AAV9 + 8 x AAV9 + 10 AAV9 +11 AAV9 + 12 AAV10 AAV10 + 8 AAV10 + 9 x AAV10 + 11 AAV10 + 12 AAV11AAV11 + 8 AAV11 + 9 AAV11 + 10 x AAV11 + 12 AAV12 AAV12 + 8 AAV12 + 9AAV12 + 10 AAV12 + 11 x

By including individual amino acids or regions from multiple AAVserotypes in one capsid protein, capsid proteins that have multipledesired properties that are separately derived from the multiple AAVserotypes may be obtained.

The genomic sequences of various serotypes of AAV and the autonomousparvoviruses, as well as the sequences of the native terminal repeats(TRs), Rep proteins, and capsid subunits are known in the art. Suchsequences may be found in the literature or in public databases such asGenBank. See, e.g., GenBank Accession Numbers NC_002077, NC_001401,NC_001729, NC_001863, NC_001829, NC_001 862, NC_000883, NC_001701,NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705,AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226,AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579; thedisclosures of which are incorporated by reference herein for teachingparvovirus and AAV nucleic acid and amino acid sequences. See also,e.g., Srivistava et al., (1983) J. Virology 45:555; Chiorini et al,(1998) J Virology 71:6823; Chiorini et al., (1999) J. Virology 73: 1309;Bantel-Schaal et al., (1999) J Virology 73:939; Xiao et al, (1999) JVirology 73:3994; Muramatsu et al., (1996) Virology 221:208; Shade etal, (1986) J. Virol. 58:921; Gao et al, (2002) Proc. Nat. Acad. Sci. USA99:11854; Moris et al, (2004) Virology 33:375-383; international patentpublications WO 00/28061, WO 99/61601, WO 98/11244; and U.S. Pat. No.6,156,303; the disclosures of which are incorporated by reference hereinfor teaching parvovirus and AAV nucleic acid and amino acid sequences.See also Table 2. The capsid structures of autonomous parvoviruses andAAV are described in more detail in BERNARD N. FIELDS et al., VIROLOGY,volume 2, chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). Seealso, description of the crystal structure of AAV2 (Xie et al., (2002)Proc. Nat. Acad. Sci. 99: 10405-10), AAV9 (DiMattia et al., (2012) J.Virol. 86:6947-6958), AAV8 (Nam et al, (2007) J. Virol. 81:12260-12271), AAV6 (Ng et al., (2010) J. Virol. 84:12945-12957), AAV5(Govindasamy et al. (2013) J. Virol. 87, 11187-11199), AAV4 (Govindasamyet al. (2006) J. Virol. 80:11556-11570), AAV3B (Lerch et al., (2010)Virology 403:26-36), BPV (Kailasan et al., (2015) J. Virol.89:2603-2614) and CPV (Xie et al, (1996) J. Mol. Biol. 6:497-520 andTsao et al, (1991) Science 251:1456-64).

TABLE 2 GenBank GenBank GenBank Accession Accession Accession NumberNumber Number Complete Clade C Rh57 AY530569 Genomes Adeno-associatedNC_002077, Hu9 AY530629 Rh50 AY530563 virus 1 AF063497 Adeno-associatedNC_001401 Hu10 AY530576 Rh49 AY530562 virus 2 Adeno-associated NC_001729Hu11 AY530577 Hu39 AY530601 virus 3 Adeno-associated NC_001863 Hu53AY530615 Rh58 AY530570 virus 3B Adeno-associated NC_001829 Hu55 AY530617Rh61 AY530572 virus 4 Adeno-associated Y18065, Hu54 AY530616 Rh52AY530565 virus 5 AF085716 Adeno-associated NC_001862 Hu7 AY530628 Rh53AY530566 virus 6 Avian AAV ATCC AY186198, Hu18 AY530583 Rh51 AY530564VR-865 AY629583, NC_004828 Avian AAV strain NC_006263, Hu15 AY530580Rh64 AY530574 DA-1 AY629583 Bovine AAV NC_005889, Hu16 AY530581 Rh43AY530560 AY388617, AAR26465 AAV11 AAT46339, Hu25 AY530591 AAV8 AF513852AY631966 AAV12 ABI16639, Hu60 AY530622 Rh8 AY242997 DQ813647 Clade A Ch5AY243021 Rh1 AY530556 AAV1 NC_002077, Hu3 AY530595 Clade F AF063497 AAV6NC_001862 Hu1 AY530575 Hu14 AY530579 (AAV9) Hu.48 AY530611 Hu4 AY530602Hu31 AY530596 Hu 43 AY530606 Hu2 AY530585 Hu32 AY530597 Hu 44 AY530607Hu61 AY530623 HSC1 MI332400.1 Hu 46 AY530609 Clade D HSC2 MI332401.1Clade B Rh62 AY530573 HSC3 MI332402.1 Hu. 19 AY530584 Rh48 AY530561 HSC4MI332403.1 Hu. 20 AY530586 Rh54 AY530567 HSC5 MI332405.1 Hu 23 AY530589Rh55 AY530568 HSC6 MI332404.1 Hu22 AY530588 Cy2 AY243020 HSC7 MI332407.1Hu24 AY530590 AAV7 AF513851 HSC8 MI332408.1 Hu21 AY530587 Rh35 AY243000HSC9 MI332409.1 Hu27 AY530592 Rh37 AY242998 HSC11 MI332406.1 Hu28AY530593 Rh36 AY242999 HSC12 MI332410.1 Hu 29 AY530594 Cy6 AY243016HSC13 MI332411.1 Hu63 AY530624 Cy4 AY243018 HSC14 MI332412.1 Hu64AY530625 Cy3 AY243019 HSC15 MI332413.1 Hu13 AY530578 Cy5 AY243017 HSC16MI332414.1 Hu56 AY530618 Rh13 AY243013 HSC17 MI332415.1 Hu57 AY530619Clade E Hu68 Hu49 AY530612 Rh38 AY530558 Clonal Isolate Hu58 AY530620Hu66 AY530626 AAV5 Y18065, AF085716 Hu34 AY530598 Hu42 AY530605 AAV 3NC_001729 Hu35 AY530599 Hu67 AY530627 AAV 3B NC_001863 AAV2 NC_001401Hu40 AY530603 AAV4 NC_001829 Hu45 AY530608 Hu41 AY530604 Rh34 AY243001Hu47 AY530610 Hu37 AY530600 Rh33 AY243002 Hu51 AY530613 Rh40 AY530559Rh32 AY243003 Hu52 AY530614 Rh2 AY243007 Others Hu T41 AY695378 Bb1AY243023 Rh74 Hu S17 AY695376 Bb2 AY243022 Bearded Dragon AAV Hu T88AY695375 Rh10 AY243015 Snake NC_006148.1 AAV Hu T71 AY695374 Hu17AY530582 Hu T70 AY695373 Hu6 AY530621 Hu T40 AY695372 Rh25 AY530557 HuT32 AY695371 Pi2 AY530554 Hu T17 AY695370 Pi1 AY530553 Hu LG15 AY695377Pi3 AY530555

The term “tropism” as used herein refers to preferential entry of thevirus into certain cells or tissues, optionally followed by expression(e.g., transcription and, optionally, translation) of a sequence(s)carried by the viral genome in the cell, e.g., for a recombinant virus,expression of a heterologous nucleic acid(s) of interest.

Those skilled in the art will appreciate that transcription of aheterologous nucleic acid sequence from the viral genome may not beinitiated in the absence of trans-acting factors, e.g., for an induciblepromoter or otherwise regulated nucleic acid sequence. In the case of arAAV genome, gene expression from the viral genome may be from a stablyintegrated provirus, from a non-integrated episome, as well as any otherform in which the virus may take within the cell.

As used herein, “systemic tropism” and “systemic transduction” (andequivalent terms) indicate that the virus capsid or virus vector of thedisclosure exhibits tropism for or transduces, respectively, tissuesthroughout the body (e.g., brain, lung, skeletal muscle, heart, liver,kidney and/or pancreas). In embodiments, systemic transduction of muscletissues (e.g., skeletal muscle, diaphragm and cardiac muscle) isobserved. In other embodiments, systemic transduction of skeletal muscletissues achieved. For example, in particular embodiments, essentiallyall skeletal muscles throughout the body are transduced (although theefficiency of transduction may vary by muscle type). In particularembodiments, systemic transduction of limb muscles, cardiac muscle anddiaphragm muscle is achieved. Optionally, the virus capsid or virusvector is administered via a systemic route (e.g., systemic route suchas intravenously, intra-articularly or intra-lymphatically).

Alternatively, in other embodiments, the capsid or virus vector isdelivered locally (e.g., to the footpad, intramuscularly, intradermally,subcutaneously, topically). Unless indicated otherwise, “efficienttransduction” or “efficient tropism,” or similar terms, can bedetermined by reference to a suitable control (e.g., at least about 50%,about 60%, about 70%, about 80%, about 85%, about 90%, about 95% or moreof the transduction or tropism, respectively, of the control). Inparticular embodiments, the virus vector efficiently transduces or hasefficient tropism for skeletal muscle, cardiac muscle, diaphragm muscle,pancreas (including β-islet cells), spleen, the gastrointestinal tract(e.g., epithelium and/or smooth muscle), cells of the central nervoussystem, eye, lung, joint cells, and/or kidney. Suitable controls willdepend on a variety of factors including the desired tropism profile.For example, AAV8 and AAV9 are highly efficient in transducing skeletalmuscle, cardiac muscle and diaphragm muscle, but have the disadvantageof also transducing liver with high efficiency. Thus, viral vectors canbe identified that demonstrate the efficient transduction of skeletal,cardiac and/or diaphragm muscle of AAV8 or AAV9, but with a much lowertransduction efficiency for liver. Further, because the tropism profileof interest may reflect tropism toward multiple target tissues, it willbe appreciated that a suitable vector may represent some tradeoffs. Toillustrate, a virus vector of the disclosure may be less efficient thanAAV8 or AAV9 in transducing skeletal muscle, cardiac muscle and/ordiaphragm muscle, but because of low level transduction of liver, maynonetheless be very desirable.

Similarly, it can be determined if a virus “does not efficientlytransduce” or “does not have efficient tropism” for a target tissue, orsimilar terms, by reference to a suitable control. In particularembodiments, the virus vector does not efficiently transduce (i.e., hasdoes not have efficient tropism) for liver, kidney, gonads and/or germcells. In particular embodiments, undesirable transduction of tissue(s)(e.g., liver) is about 20% or less, about 10% or less, about 5% or less,about 1% or less, about 0.1% or less of the level of transduction of thedesired target tissue(s) (e.g., skeletal muscle, diaphragm muscle,cardiac muscle and/or cells of the central nervous system).

As used herein, the term “polypeptide” encompasses both peptides andproteins, unless indicated otherwise.

A “polynucleotide” is a sequence of nucleotide bases, and may be RNA,DNA or DNA-RNA hybrid sequences (including both naturally occurring andnon-naturally occurring nucleotide), but in representative embodimentsare either single or double stranded DNA sequences.

As used herein, an “isolated” polynucleotide (e.g., an “isolated DNA” oran “isolated RNA”) means a polynucleotide at least partially separatedfrom at least some of the other components of the naturally occurringorganism or virus, for example, the cell or viral structural componentsor other polypeptides or nucleic acids commonly found associated withthe polynucleotide. In representative embodiments an “isolated”nucleotide is enriched by at least about 10-fold, about 100-fold, about1000-fold, about 10,000-fold or more as compared with the startingmaterial.

Likewise, an “isolated” polypeptide means a polypeptide that is at leastpartially separated from at least some of the other components of thenaturally occurring organism or virus, for example, the cell or viralstructural components or other polypeptides or nucleic acids commonlyfound associated with the polypeptide. In representative embodiments an“isolated” polypeptide is enriched by at least about 10-fold, 100-fold,1000-fold, 10,000-fold or more as compared with the starting material.

As used herein, by “isolate” or “purify” (or grammatical equivalents) avirus vector, it is meant that the virus vector is at least partiallyseparated from at least some of the other components in the startingmaterial. In representative embodiments an “isolated” or “purified”virus vector is enriched by at least about 10-fold, about 100-fold,about 1000-fold, about 10,000-fold or more as compared with the startingmaterial.

A “therapeutic polypeptide” is a polypeptide that can alleviate, reduce,prevent, delay and/or stabilize symptoms that result from an absence ordefect in a protein in a cell or subject and/or is a polypeptide thatotherwise confers a benefit to a subject, e.g., anti-cancer effects orimprovement in transplant survivability.

By the terms “treat,” “treating” or “treatment of” (and grammaticalvariations thereof) it is meant that the severity of the subject'scondition is reduced, at least partially improved or stabilized and/orthat some alleviation, mitigation, decrease or stabilization in at leastone clinical symptom is achieved and/or there is a delay in theprogression of the disease or disorder.

The terms “prevent,” “preventing” and “prevention” (and grammaticalvariations thereof) refer to prevention and/or delay of the onset of adisease, disorder and/or a clinical symptom(s) in a subject and/or areduction in the severity of the onset of the disease, disorder and/orclinical symptom(s) relative to what would occur in the absence of themethods of the disclosure. The prevention can be complete, e.g., thetotal absence of the disease, disorder and/or clinical symptom(s). Theprevention can also be partial, such that the occurrence of the disease,disorder and/or clinical symptom(s) in the subject and/or the severityof onset is less than what would occur in the absence of the presentdisclosure.

“Therapeutically effective amount” as used herein refers to an amountthat, when administered to a subject for treating a disease, or at leastone of the clinical symptoms of a disease, is sufficient to affect suchtreatment of the disease or symptom thereof. The “therapeuticallyeffective amount” may vary depending, for example, on the disease and/orsymptoms of the disease, severity of the disease and/or symptoms of thedisease or disorder, the age, weight, and/or health of the patient to betreated, and the judgment of the prescribing physician. An appropriateamount in any given instance may be ascertained by those skilled in theart or capable of determination by routine experimentation.

As used herein, the terms “virus vector,” “vector” or “gene deliveryvector” refer to a virus (e.g., AAV) particle that functions as anucleic acid delivery vehicle, and which comprises the vector genome(e.g., viral DNA [vDNA]) packaged within a virion.

Alternatively, in some contexts, the term “vector” may be used to referto the vector genome/vDNA alone.

A “rAAV vector genome” or “rAAV genome” is an AAV genome (i.e., vDNA)that comprises one or more heterologous nucleic acid sequences. rAAVvectors generally require only the terminal repeat(s) (TR(s)) in cis togenerate virus. All other viral sequences are dispensable and may besupplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol.158:97). Typically, the rAAV vector genome will only retain the one ormore TR sequence so as to maximize the size of the transgene that can beefficiently packaged by the vector. The structural and non-structuralprotein coding sequences may be provided in trans (e.g., from a vector,such as a plasmid, or by stably integrating the sequences into apackaging cell). In embodiments, the rAAV vector genome comprises atleast one TR sequence (e.g., AAV TR sequence), optionally two TRs (e.g.,two AAV TRs), which typically will be at the 5′ and 3′ ends of thevector genome and flank the heterologous nucleic acid, but need not becontiguous thereto. The TRs can be the same or different from eachother.

The term “terminal repeat” or “TR” includes any viral terminal repeat orsynthetic sequence that forms a hairpin structure and functions as aninverted terminal repeat (i.e., mediates the desired functions such asreplication, virus packaging, integration and/or provirus rescue, andthe like). The TR can be an AAV TR or a non-AAV TR. For example, anon-AAV TR sequence such as those of other parvoviruses (e.g., canineparvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or anyother suitable virus sequence (e.g., the SV40 hairpin that serves as theorigin of SV40 replication) can be used as a TR, which can further bemodified by truncation, substitution, deletion, insertion and/oraddition. Further, the TR can be partially or completely synthetic, suchas the “double-D sequence” as described in U.S. Pat. No. 5,478,745 toSamulski et al.

An “AAV terminal repeat” or “AAV TR” may be from any AAV, including butnot limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 orany other AAV now known or later discovered (see, e.g., Table 2). An AAVterminal repeat need not have the native terminal repeat sequence (e.g.,a native AAV TR sequence may be altered by insertion, deletion,truncation and/or missense mutations), as long as the terminal repeatmediates the desired functions, e.g., replication, virus packaging,integration, and/or provirus rescue, and the like.

The virus vectors of the disclosure can further be “targeted” virusvectors (e.g., having a directed tropism) and/or a “hybrid” parvovirus(i.e., in which the viral TRs and viral capsid are from differentparvoviruses) as described in international patent publicationWO00/28004 and Chao et al, (2000) Molecular Therapy 2:619.

The virus vectors of the disclosure can further be duplexed parvovirusparticles as described in international patent publication WO 01/92551(the disclosure of which is incorporated herein by reference in itsentirety). Thus, in some embodiments, double stranded (duplex) genomescan be packaged into the virus capsids of the disclosure.

Further, the viral capsid or genomic elements can contain othermodifications, including insertions, deletions and/or substitutions.

As used herein, the term “amino acid” encompasses any naturallyoccurring amino acid, modified forms thereof, and synthetic amino acids.

Naturally occurring, levorotatory (L-) amino acids are shown in Table 3.

TABLE 3 Amino acid residues and abbreviations. Abbreviation Amino AcidResidue Three-Letter Code One-Letter Code Alanine Ala A Arginine Arg RAsparagine Asn N Aspartic acid (Aspartate) Asp D Cysteine Cys CGlutamine Gln Q Glutamic acid (Glutamate) Glu E Glycine Gly G HistidineHis H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met MPhenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

Alternatively, the amino acid can be a modified amino acid residue(nonlimiting examples are shown in Table 4) and/or can be an amino acidthat is modified by post-translation modification (e.g., acetylation,amidation, formylation, hydroxylation, methylation, phosphorylation orsulfatation).

TABLE 4 Modified Amino Acid Residues Modified Amino Acid ResidueAbbreviation Amino Acid Residue Derivatives 2-Aminoadipic acid Aad3-Aminoadipic acid bAad beta-Alanine, beta-Aminoproprionic acid bAla2-Aminobutyric acid Abu 4-Aminobutyric acid, Piperidinic acid 4Abu6-Aminocaproic acid Acp 2-Aminoheptanoic acid Ahe 2-Aminoisobutyric acidAib 3-Aminoisobutyric acid bAib 2-Aminopimelic acid Apm t-butylalaninet-BuA Citrulline Cit Cyclohexylalanine Cha 2,4-Diaminobutyric acid DbuDesmosine Des 2,21-Diaminopimelic acid Dpm 2,3-Diaminoproprionic acidDpr N-Ethylglycine EtGly N-Ethylasparagine EtAsn Homoarginine hArgHomocysteine hCys Homoserine hSer Hydroxylysine Hyl Allo-HydroxylysineaHyl 3-Hydroxyproline 3Hyp 4-Hydroxyproline 4Hyp Isodesmosine Ideallo-lsoleucine alle Methionine sulfoxide MSO N-Methylglycine, sarcosineMeGly N-Methyl isoleucine Melle 6-N-Methyllysine MeLys N-MethylvalineMeVal 2-Naphthylalanine 2-Nal Norvaline Nva Norleucine Nle Ornithine Orn4-Chlorophenylalanine Phe(4-C1) 2-Fluorophenylalanine Phe(2-F)3-Fluorophenylalanine Phe(3-F) 4-Fluorophenylalanine Phe(4-F)Phenylglycine Phg Beta-2-thienylalanine Thi

Further, the non-naturally occurring amino acid can be an “unnatural”amino acid (as described by Wang et al., Annu Rev Biophys Biomol Struct.35:225-49 (2006)). These unnatural amino acids can advantageously beused to chemically link molecules of interest to the AAV capsid protein.

Modified AAV Capsid Proteins and Virus Capsids and Virus VectorsComprising the Same.

The present disclosure provides AAV capsid proteins (VP1, VP2 and/orVP3) comprising a modification (e.g., a substitution and/or a deletion)in the amino acid sequence and virus capsids and virus vectorscomprising the modified AAV capsid protein. The inventors havediscovered that the modifications described herein can confer one ormore desirable properties to virus vectors comprising the modified AAVcapsid protein including without limitation, selective transduction to atarget tissue of interest. In some embodiments, the target tissue ofinterest may be an ophthalmic tissue, such as the retinal pigmentepithelium (RPE). Thus, the present disclosure addresses some of thelimitations associated with conventional AAV vectors.

As used herein, a “mutation” or “modification” in an amino acid sequencecan include substitutions, insertions and/or deletions, each of whichcan involve one, two, three, four, five, six, seven, eight, nine, ten ormore amino acids. In particular embodiments, the modification is asubstitution.

The modified virus capsid proteins of the invention can be but are notlimited to AAV capsid proteins in which amino acids from one AAV capsidprotein are substituted into another AAV capsid protein, and thesubstituted and/or inserted amino acids can be from any source, and canfurther be natural or partially or completely synthetic.

In some embodiments, the present disclosure provides an adeno-associatedvirus (AAV) capsid protein, comprising one or more amino acidsubstitutions, wherein the one or more substitutions modify one or moresurface-exposed loops on the AAV capsid protein. The modification of theone or more surface-exposed loops results in enhanced transduction to atarget tissue. The target tissue may be an ophthalmic tissue, such asthe retinal pigment epithelium (RPE). The one or more amino acidsubstitutions can be in one or more surface-exposed loops identified bypeptide epitope mapping and/or cryo-electron microscopy studies.

The capsid proteins of this disclosure are modified to produce an AAVcapsid that is present in an AAV virus particle or AAV virus vector thathas a phenotype of enhanced transduction with respect to a target tissueof interest (e.g., an ophthalmic tissue such as the retinal pigmentepithelium). The AAV virus particle or vector of this disclosure canalso have a phenotype of evading neutralizing antibodies.

In some embodiments, the one or more substitutions of the one or moresurface-exposed loops can introduce one or more surface-exposed loopsfrom a capsid protein of a first AAV serotype into the capsid protein ofa second AAV serotype that is different from said first AAV serotype.

The AAV capsid protein of this disclosure can be a capsid protein of anAAV serotype selected from AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8, AAVrh.10, AAVrh.32.33,AAVrh74, bovine AAV, avian AAV or any other AAV now known or lateridentified. In some embodiments, the AAV capsid protein is chimeric.

In some embodiments, the amino acid substitution replaces any six aminoacids in an AAV capsid protein from any one of the serotypes listed inthe preceding paragraph. For example, the amino acid substitution mayreplace the following amino acids (VP1 numbering) in an AAV capsidprotein from any one of the serotypes listed in the preceding paragraph:397-402, 403-408, 409-414, 415-420, 421-426, 427-432, 433-438, 439-444,445-450, 451-456, 457-462, 463-468, 469-474, 475-480, 481-486, 487-492,493-489, 490-495, 496-501, 500-505, 506-510, 511-517, 523-528, 529-534,535-540, 541-546, 547-552, 553-558, 559-560, 561-565, 566-571, 572-577,578-583, 584-589, 590-595, 596-601, 602-607, 608-613, 614-619, 620-625,626-631, 632-637, 638-642, 643-648, 649-654, 655-670, 617-676, 677-682,683-688, 689-694, 695-700, 701-706.

In some embodiments, the AAV capsid protein comprises one or more aminoacid substitutions that do not occur in the capsid sequence, wherein theamino acid substitutions are selected from the sequences listed in Table5.

TABLE 5 AMINO ACID SUBSTITUTIONS Sequence Substitution SEQ ID NO. KVRDLF 14 RVLALR  15 RVHALR  16 RVHSLR  17 GVGVLP  18 FVNALN  19 IVRSLN  20HVLRLN  21 RVLALQ  22 RVRGLR 187 KVRTLR 188 MVGNLV 189 RVLGLR 190 KVAGLC191 IVRPLV 192 KVRGLA 193 RVRGLG 194

In some embodiments, the amino acid substitution replaces the aminoacids in the region corresponding to amino acid positions 591-596 of SEQID NO. 11 or amino acids 590-595 of SEQ ID NO. 12.

In some embodiments, the AAV capsid comprises the amino acid sequence ofany one of SEQ ID NO: 11-12, 23-49, or 195-254. In some embodiments, thecapsid comprises an amino acid sequence that has at least 90%, at least95%, at least 96%, at least 97%, at least 98% or at least 99% sequencehomology with any one of SEQ ID NO: 11-12, 23-49, or 195-254.

In some embodiments, a recombinant capsid protein has a sequence that isat least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% or 100% identical to any one of SEQ ID NO: 32, 35, 37, or 40.

In some embodiments, an AAV capsid protein of SEQ ID NO: 4 comprises oneor more of the following amino acid substitutions: K492E, K503E, N585S,T590K, T590R, T590G, T590F, T5901, T590H, T590M, D592R, D592L, D592H,D592G, D592N, D592A, R593D, R593A, R593S, R593V, R593R, R593G, R593T,R593N, R593P, T595F, T595R, T595P, T595N, T595Q, T595V, T595C, T595A,T595G.

In some embodiments, an AAV capsid protein of SEQ ID NO: 11 comprisesone or more of the following amino acid substitutions: T591K, T591R,T591G, T591F, T591 I, T591H, T591M, D593R, D593L, D593H, D593G, D593N,D593A, R594D, R594A, R594S, R594V, R594R, R594G, R594T, R594N, R594P,T596F, T596R, T596P, T596N, T596Q, T596V, T596C, T596A, T596G.

In some embodiments, an AAV capsid protein of SEQ ID NO: 12 comprisesone or more of the following amino acid substitutions: T590K, T590R,T590G, T590F, T5901, T590H, T590M, D592R, D592L, D592H, D592G, D592N,D592A, R593D, R593A, R593S, R593V, R593R, R593G, R593T, R593N, R593P,T595F, T595R, T595P, T595N, T595Q, T595V, T595C, T595A, T595G.

In some embodiments, the AAV capsid protein comprises a substitutioncomprising a sequence of six amino acids (X¹-X²-X³-X⁴-X⁵-X⁶) that doesnot occur in the native AAV capsid protein, wherein X² is V and X⁵ is L(SEQ ID NO: 186). In some embodiments, X¹ is not T, X³ is not D, X⁴ isnot R, and X⁶ is not T (SEQ ID NO: 255). In some embodiments, X¹ is K,G, F, I, H, or R (SEQ ID NO: 256). In some embodiments, X³ is R, L, H,G, or N (SEQ ID NO: 257). In some embodiments, X⁴ is D, A, S, V, or R(SEQ ID NO: 258). In some embodiments, X⁶ is F, R, P, N, or Q (SEQ IDNO: 259). In some embodiments, X¹ is K, X³ is R, X⁴ is D, and X⁶ is F(SEQ ID NO: 14). In some embodiments, X¹ is R, X³ is L, X⁴ is A, and X⁶is R (SEQ ID NO: 15). In some embodiments, X¹ is R, X³ is H, X⁴ is A,and X⁶ is R (SEQ ID NO: 16). In some embodiments, X¹ is R, X³ is H, X⁴is S, and X⁶ is R (SEQ ID NO: 17). In some embodiments, X¹ is G, X³ isG, X⁴ is V, and X⁶ is P (SEQ ID NO: 18). In some embodiments, X¹ is F,X³ is N, X⁴ is A, and X⁶ is N (SEQ ID NO: 19). In some embodiments, X¹is I, X³ is R, X⁴ is S, and X⁶ is N (SEQ ID NO: 20). In someembodiments, X¹ is H, X³ is L, X⁴ is R, and X⁶ is N (SEQ ID NO: 21). Insome embodiments, X¹ is R, X³ is L, X⁴ is A, and X⁶ is Q (SEQ ID NO:22). In some embodiments, X¹ is R, X³ is L, X⁴ is A, and X⁶ is Q (SEQ IDNO: 187). In some embodiments, X¹ is K, X³ is R, X⁴ is T, and X⁶ is R(SEQ ID NO: 188). In some embodiments, X¹ is M, X³ is G, X⁴ is N, and X⁶is V (SEQ ID NO: 189). In some embodiments, X¹ is R, X³ is L, X⁴ is G,and X⁶ is R (SEQ ID NO: 190). In some embodiments, X¹ is K, X³ is A, X⁴is G, and X⁶ is C (SEQ ID NO: 191). In some embodiments, X¹ is I, X³ isV, X⁴ is R, and X⁶ is V (SEQ ID NO: 192). In some embodiments, X¹ is K,X³ is R, X⁴ is G, and X⁶ is A (SEQ ID NO: 193). In some embodiments, X¹is R, X³ is R, X⁴ is G, and X⁶ is G (SEQ ID NO: 194).

The present disclosure also provides a nucleotide sequence, or anexpression vector comprising the same, that encodes one or more of theAAV capsid proteins of the disclosure. The nucleotide sequence may be aDNA sequence or an RNA sequence. The present disclosure also provides acell that comprises one or more nucleotide sequences or expressionvectors of the disclosure.

Also provided is an AAV capsid comprising an AAV capsid protein of thisdisclosure. Further provided herein is a viral vector comprising an AAVcapsid of this disclosure as well as a composition comprising the AAVcapsid protein, AAV capsid and/or viral vector of this disclosure in apharmaceutically acceptable carrier.

In some embodiments, modification of the one or more surface-exposedloops results in an enhanced transduction profile with respect to atarget tissue of interest, for example an ophthalmic tissue such as theretinal pigment epithelium (RPE). In some embodiments, modification ofthe one or more surface-exposed loops results in inhibition of bindingby an antibody to one or more antigenic sites on the AAV capsid protein.In some embodiments, modification of the one or more surface-exposedloops results in inhibition of neutralization of infectivity of a virusparticle comprising the AAV capsid protein.

As described herein, the nucleic acid and amino acid sequences of thecapsid proteins from a number of AAV are known in the art. Thus, theamino acids “corresponding” to amino acid positions of the native AAVcapsid protein can be readily determined for any other AAV (e.g., byusing sequence alignments).

The disclosure contemplates that the modified capsid proteins can beproduced by modifying the capsid protein of any AAV now known or laterdiscovered.

Further, the AAV capsid protein that is to be modified can be anaturally occurring AAV capsid protein (e.g., an AAV2, AAV3a or 3b,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11 capsid protein or anyof the AAV shown in Table 2) but is not so limited. Those skilled in theart will understand that a variety of manipulations to the AAV capsidproteins are known in the art and the disclosure is not limited tomodifications of naturally occurring AAV capsid proteins. For example,the capsid protein to be modified may already have alterations ascompared with naturally occurring AAV (e.g., is derived from a naturallyoccurring AAV capsid protein, e.g., AAV2, AAV3a, AAV3b, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 or any other AAV now knownor later discovered). In some embodiments, the capsid protein to bemodified may be a chimeric capsid protein. In some embodiments, thecapsid protein to be modified may be an engineered AAV, such as AAV2i8,AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV PHP.B. In some embodiments, thecapsid protein to be modified may have the sequence of SEQ ID NO:11 orSEQ ID NO:12.

Thus, in particular embodiments, the AAV capsid protein to be modifiedcan be derived from a naturally occurring AAV but further comprises oneor more foreign sequences (e.g., that are exogenous to the native virus)that are inserted and/or substituted into the capsid protein and/or hasbeen altered by deletion of one or more amino acids.

Accordingly, when referring herein to a specific AAV capsid protein(e.g., an AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or AAV11capsid protein or a capsid protein from any of the AAV shown in Table 2,etc.), it is intended to encompass the native capsid protein as well ascapsid proteins that have alterations other than the modifications ofthe disclosure. Such alterations include substitutions, insertionsand/or deletions. In particular embodiments, the capsid proteincomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20, less than 20, less than 30, less than 40, less than 50, lessthan 60, or less than 70 amino acids inserted therein (other than theinsertions of the present disclosure) as compared with the native AAVcapsid protein sequence. In embodiments, the capsid protein comprises 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20,less than 20, less than 30, less than 40, less than 50, less than 60, orless than 70 amino acid substitutions (other than the amino acidsubstitutions according to the present disclosure) as compared with thenative AAV capsid protein sequence, in embodiments of the disclosure,the capsid protein comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, less than 20, less than30, less than 40, less than 50, less than 60, or less than 70 aminoacids (other than the amino acid deletions of the disclosure) ascompared with the native AAV capsid protein sequence.

In particular embodiments, the AAV capsid protein has the native AAVcapsid protein sequence or has an amino acid sequence that is at leastabout 90%, about 95%, about 97%, about 98% or about 99% similar oridentical to a native AAV capsid protein sequence. For example, inparticular embodiments, an “AAV4” capsid protein encompasses the nativeAAV4 capsid protein sequence as well as sequences that are at leastabout 90%, about 95%, about 97%, about 98% or about 99% similar oridentical to the native AAV4 capsid protein sequence.

Methods of determining sequence similarity or identity between two ormore amino acid sequences are known in the art. Sequence similarity oridentity may be determined using standard techniques known in the art,including, but not limited to, the local sequence identity algorithm ofSmith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequenceidentity alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48,443(1970), by the search for similarity method of Pearson & Lipman, Proc.Natl. Acad. Sci. USA 85,2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive,Madison, Wis.), the Best Fit sequence program described by Devereux etal., Nucl. Acid Res. 12, 387-395 (1984), or by inspection.

Another suitable algorithm is the BLAST algorithm, described in Altschulet al., J Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl.Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST programis the WU-BLAST-2 program which was obtained from Altschul et al.,Methods in Enzymology, 266, 460-480 (1996);

http://blast.wustl/edu/blast/README.html. WU-BLAST-2 uses several searchparameters, which are optionally set to the default values. Theparameters are dynamic values and are established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity. Further, an additional useful algorithm is gappedBLAST as reported by Altschul et al, (1997) Nucleic Acids Res. 25,3389-3402.

The disclosure also provides a virus capsid comprising, consistingessentially of, or consisting of the modified AAV capsid protein of thedisclosure. In particular embodiments, the virus capsid is a parvoviruscapsid, which may further be an autonomous parvovirus capsid or adependovirus capsid. Optionally, the virus capsid is an AAV capsid. Inparticular embodiments, the AAV capsid is an AAV1, AAV2, AAV3a, AAV3b,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8,AAVrh10, AAVrh32.33, bovine AAV capsid, avian AAV capsid or any otherAAV now known or later identified. A nonlimiting list of AAV serotypesis shown in Table 2. An AAV capsid of this disclosure can be any AAVserotype listed in Table 2 or derived from any of the foregoing by oneor more insertions, substitutions and/or deletions.

The modified virus capsids can be used as “capsid vehicles,” as has beendescribed, for example, in U.S. Pat. No. 5,863,541. Molecules that canbe packaged by the modified virus capsid and transferred into a cellinclude heterologous DNA, RNA, polypeptides, small organic molecules,metals, or combinations of the same.

Heterologous molecules are defined as those that are not naturally foundin an AAV infection, e.g., those not encoded by a wild-type AAV genome.Further, therapeutically useful molecules can be associated with theoutside of the chimeric virus capsid for transfer of the molecules intohost target cells. Such associated molecules can include DNA, RNA, smallorganic molecules, metals, carbohydrates, lipids and/or polypeptides. Inone embodiment of the disclosure the therapeutically useful molecule iscovalently linked (i.e., conjugated or chemically coupled) to the capsidproteins. Methods of covalently linking molecules are known by thoseskilled in the art.

The modified virus capsids of the disclosure also find use in raisingantibodies against the novel capsid structures. As a furtheralternative, an exogenous amino acid sequence may be inserted into themodified virus capsid for antigen presentation to a cell, e.g., foradministration to a subject to produce an immune response to theexogenous amino acid sequence.

In other embodiments, the virus capsids can be administered to blockcertain cellular sites prior to and/or concurrently with (e.g., withinminutes or hours of each other) administration of a virus vectordelivering a nucleic acid encoding a polypeptide or functional RNA ofinterest. For example, the capsids of the disclosure can be delivered toblock cellular receptors on liver cells and a delivery vector can beadministered subsequently or concurrently, which may reduce transductionof liver cells, and enhance transduction of other targets (e.g.,skeletal, cardiac and/or diaphragm muscle).

According to representative embodiments, modified virus capsids can beadministered to a subject prior to and/or concurrently with a modifiedvirus vector according to the present disclosure. Further, thedisclosure provides compositions and pharmaceutical formulationscomprising the modified virus capsids; optionally, the composition alsocomprises a modified virus vector of the disclosure.

The disclosure also provides nucleic acids (optionally, isolated nucleicacids) encoding the modified virus capsids and capsid proteins of thedisclosure. Further provided are vectors comprising the nucleic acids,and cells (in vivo or in culture) comprising the nucleic acids and/orvectors of the disclosure. As one example, the present disclosureprovides a virus vector comprising: (a) a modified AAV capsid of thisdisclosure; and (b) a nucleic acid comprising at least one terminalrepeat sequence, wherein the nucleic acid is encapsidated by the AAVcapsid.

Other suitable vectors include without limitation viral vectors (e.g.,adenovirus, AAV, herpesvirus, vaccinia, poxviruses, baculoviruses, andthe like), plasmids, phage, YACs, BACs, and the like. Such nucleicacids, vectors and cells can be used, for example, as reagents (e.g.,helper packaging constructs or packaging cells) for the production ofmodified virus capsids or virus vectors as described herein.

Virus capsids according to the disclosure can be produced using anymethod known in the art, e.g., by expression from a baculovirus (Brownet al., (1994) Virology 198:477-488).

The modifications to the AAV capsid protein according to the presentdisclosure are “selective” modifications. This approach is in contrastto previous work with whole subunit or large domain swaps between AAVserotypes (see, e.g., international patent publication WO 00/28004 andHauck et al., (2003) J. Virology 77:2768-2774). In particularembodiments, a “selective” modification results in the insertion and/orsubstitution and/or deletion of less than or equal to about 20, about18, about 15, about 12, about 10, about 9, about 8, about 7, about 6,about 5, about 4 or about 3 contiguous amino acids.

The modified capsid proteins and capsids of the disclosure can furthercomprise any other modification, now known or later identified.

For example, the AAV capsid proteins and virus capsids of the disclosurecan be chimeric in that they can comprise all or a portion of a capsidsubunit from another virus, optionally another parvovirus or AAV, e.g.,as described in international patent publication WO 00/28004.

In some embodiments of this disclosure, the virus capsid can be atargeted virus capsid, comprising a targeting sequence (e.g.,substituted or inserted in the viral capsid) that directs the viruscapsid to interact with cell-surface molecules present on desired targettissue(s) (see, e.g., International patent publication WO 00/28004 andHauck et al., (2003) J. Virology 77:2768-2774); Shi et al., Human GeneTherapy 17:353-361 (2006) [describing insertion of the integrin receptorbinding motif RGD at positions 520 and/or 584 of the AAV capsidsubunit]; and U.S. Pat. No. 7,314,912 [describing insertion of the PIpeptide containing an RGD motif following amino acid positions 447, 534,573 and 587 of the AAV2 capsid subunit]). Other positions within the AAVcapsid subunit that tolerate insertions are known in the art (e.g.,positions 449 and 588 described by Grifman et al., Molecular Therapy3:964-975 (2001)).

For example, a virus capsid of this disclosure may have relativelyinefficient tropism toward certain target tissues of interest (e.g.,liver, skeletal muscle, heart, diaphragm muscle, kidney, brain, stomach,intestines, skin, endothelial cells, and/or lungs). A targeting sequencecan advantageously be incorporated into these low-transduction vectorsto thereby confer to the virus capsid a desired tropism and, optionally,selective tropism for particular tissue(s). AAV capsid proteins, capsidsand vectors comprising targeting sequences are described, for example ininternational patent publication WO 00/28004. As another example, one ormore non-naturally occurring amino acids as described by Wang et al.,Annu Rev Biophys Biomol Struct. 35:225-49 (2006)) can be incorporatedinto an AAV capsid subunit of this disclosure at an orthogonal site as ameans of redirecting a low-transduction vector to desired targettissue(s). These unnatural amino acids can advantageously be used tochemically link molecules of interest to the AAV capsid proteinincluding without limitation: glycans (mannose-dendritic celltargeting); RGD, bombesin or a neuropeptide for targeted delivery tospecific cancer cell types; RNA aptamers or peptides selected from phagedisplay targeted to specific cell surface receptors such as growthfactor receptors, integrins, and the like.

Methods of chemically modifying amino acids are known in the art (see,e.g., Greg T. Hermanson, Bioconjugate Techniques, 1st edition, AcademicPress, 1996).

In some embodiments, the targeting sequence may be a virus capsidsequence (e.g., an autonomous parvovirus capsid sequence, AAV capsidsequence, or any other viral capsid sequence) that directs infection toa particular cell type(s).

As another nonlimiting example, a heparin or heparan sulfate bindingdomain (e.g., the respiratory syncytial virus heparin binding domain)may be inserted or substituted into a capsid subunit that does nottypically bind HS receptors (e.g., AAV4, AAV5) to confer heparin and/orheparan sulfate binding to the resulting mutant.

B19 infects primary erythroid progenitor cells using globoside as itsreceptor (Brown et al, (1993) Science 262: 114). The structure of B19has been determined to 8 Å resolution (Agbandje-McKenna et al, (1994)Virology 203: 106). The region of the B19 capsid that binds to globosidehas been mapped between amino acids 399-406 (Chapman et al, (1993)Virology 194:419), a looped out region between β-barrel structures E andF (Chipman et al, (1996) Proc. Nat. Acad. Sci. USA 93:7502).Accordingly, the globoside receptor binding domain of the B19 capsid maybe substituted into an AAV capsid protein of this disclosure to target avirus capsid or virus vector comprising the same to erythroid cells.

In some embodiments, the exogenous targeting sequence may be any aminoacid sequence encoding a peptide that alters the tropism of a viruscapsid or virus vector comprising the modified AAV capsid protein. Inparticular embodiments, the targeting peptide or protein may benaturally occurring or, alternately, completely or partially synthetic.Exemplary targeting sequences include ligands and other peptides thatbind to cell surface receptors and glycoproteins, such as ROD peptidesequences, bradykinin, hormones, peptide growth factors (e.g., epidermalgrowth factor, nerve growth factor, fibroblast growth factor,platelet-derived growth factor, insulin-like growth factors I and II,etc.), cytokines, melanocyte stimulating hormone (e.g., α, β or γ),neuropeptides and endorphins, and the like, and fragments thereof thatretain the ability to target cells to their cognate receptors. Otherillustrative peptides and proteins include substance P, keratinocytegrowth factor, neuropeptide Y, gastrin releasing peptide, interleukin 2,hen egg white lysozyme, erythropoietin, gonadolibcrin, corticostatin,8-endorphin, leu-enkephalin, rimorphin, alpha-neo-enkephalin,angiotensin, pneumadin, vasoactive intestinal peptide, neurotensin,motilin, and fragments thereof as described above. As yet a furtheralternative, the binding domain from a toxin (e.g., tetanus toxin orsnake toxins, such as alpha-bungarotoxin, and the like) can besubstituted into the capsid protein as a targeting sequence. In a yetfurther representative embodiment, the AAV capsid protein can bemodified by substitution of a “nonclassical” import/export signalpeptide (e.g., fibroblast growth factor-1 and -2, interleukin 1, HIV-1Tat protein, herpes virus VP22 protein, and the like) as described byCleves (Current Biology 7:R318 (1997)) into the AAV capsid protein. Alsoencompassed are peptide motifs that direct uptake by specific cells,e.g., a FVFLP (SEQ ID NO: 50) peptide motif triggers uptake by livercells.

Phage display techniques, as well as other techniques known in the art,may be used to identify peptides that recognize any cell type ofinterest.

The targeting sequence may encode any peptide that targets to a cellsurface binding site, including receptors (e.g., protein, carbohydrate,glycoprotein or proteoglycan). Examples of cell surface binding sitesinclude, but are not limited to, heparan sulfate, chondroitin sulfate,and other glycosaminoglycans, sialic acid moieties found on mucins,glycoproteins, and gangliosides, MHC 1 glycoproteins, carbohydratecomponents found on membrane glycoproteins, including, mannose,N-acetyl-galactosamine, N-acetyl-glucosamine, fucose, galactose, and thelike.

In particular embodiments, a heparan sulfate (HS) or heparin bindingdomain is substituted into the virus capsid (for example, in an AAVcapsid that otherwise does not bind to HS or heparin). It is known inthe art that HS/heparin binding is mediated by a “basic patch” that isrich in arginines and/or lysines. In exemplary embodiments, a sequencefollowing the motif BXXB (SEQ ID NO: 51), where “B” is a basic residueand X is neutral and/or hydrophobic can be employed. As a non-limitingexample, BXXB can be RGNR (SEQ ID NO: 52). As another non-limitingexample, BXXB is substituted for amino acid positions 262 through 265 inthe native AAV2 capsid protein or at the corresponding position(s) inthe capsid protein of another AAV serotype.

Table 6 shows other non-limiting examples of suitable targetingsequences.

TABLE 6 SUITABLE TARGETING SEQUENCES Sequence NO SEQ ID ReferenceNSVRDL(G/S)  53 Muller et al., Nature Biotechnology 21: 1040-1046 (2003)PRSVTVP  54 Muller et al., Nature Biotechnology 21: 1040-1046 (2003)NSVSSX(S/A)  55 Muller et al., Nature Biotechnology 21: 1040-1046 (2003)NGRAHA  56 Grifman et al., Molecular Therapy 3:964-975 (2001) QPEHSST 57 Work et al., Molecular Therapy 13:683-693 (2006) VNTANST  58Work et al., Molecular Therapy 13:683-693 (2006) HGPMQS  59Work et al., Molecular Therapy 13:683-693 (2006) PHKPPLA  60Work et al., Molecular Therapy 13:683-693 (2006) IKNNEMW  61Work et al., Molecular Therapy 13:683-693 (2006) RNLDTPM  62Work et al., Molecular Therapy 13:683-693 (2006) VDSHRQS  63Work et al., Molecular Therapy 13:683-693 (2006) YDSKTKT  64Work et al., Molecular Therapy 13:683-693 (2006) SQLPHQK  65Work et al., Molecular Therapy 13:683-693 (2006) STMQQNT  66Work et al., Molecular Therapy 13:683-693 (2006) TERYMTQ  67Work et al., Molecular Therapy 13:683-693 (2006) QPEHSST  68Work et al., Molecular Therapy 13:683-693 (2006) DASLSTS  69Work et al., Molecular Therapy 13:683-693 (2006) DLPNKT  70Work et al., Molecular Therapy 13:683-693 (2006) DLTAARL  71Work et al., Molecular Therapy 13:683-693 (2006) EPHQFNY  72Work et al., Molecular Therapy 13:683-693 (2006) EPQSNHT  73Work et al., Molecular Therapy 13:683-693 (2006) MSSWPSQ  74Work et al., Molecular Therapy 13:683-693 (2006) NPKHNAT  75Work et al., Molecular Therapy 13:683-693 (2006) PDGMRTT  76Work et al., Molecular Therapy 13:683-693 (2006) PNNNKTT  77Work et al., Molecular Therapy 13:683-693 (2006) QSTTHDS  78Work et al., Molecular Therapy 13:683-693 (2006) TGSKQKQ  79Work et al., Molecular Therapy 13:683-693 (2006) SLKHQAL  80Work et al., Molecular Therapy 13:683-693 (2006) SPIDGEQ  81Work et al., Molecular Therapy 13:683-693 (2006) WIFPWIQL  82Hajitou et al., TCM 16:80-88 (2006) CDCRGDCFC  83Hajitou et al., TCM 16:80-88 (2006) CNGRC  84Hajitou et al., TCM 16:80-88 (2006) CPRECES  85Hajitou et al., TCM 16:80-88 (2006) CTTHWGFTLC  86Hajitou et al., TCM 16:80-88 (2006) CGRRAGGSC  87Hajitou et al., TCM 16:80-88 (2006) CKGGRAKDC  88Hajitou et al., TCM 16:80-88 (2006) CVPELGHEC  89Hajitou et al., TCM 16:80-88 (2006) CRRETAWAK  90Koivunen et al., J. Nucl. Med. 40:883-888 (1999) VSWFSHRYSPFAV  91Koivunen et al., J. Nucl. Med. 40:883-888 (1999) S GYRDGYAGPILYN  92Koivunen et al., J. Nucl. Med. 40:883-888 (1999) XXXY*XXX  93Koivunen et al., J. Nucl. Med. 40:883-888 (1999) Y*E/MNW  94Koivunen et al., J. Nucl. Med. 40:883-888 (1999) RPLPPLP  95Koivunen et al., J. Nucl. Med. 40:883-888 (1999) APPLPPR  96Koivunen et al., J. Nucl. Med. 40:883-888 (1999) DVFYPYPYASGS  97Koivunen et al., J. Nucl. Med. 40:883-888 (1999) MYWYPY  98Koivunen et al., J. Nucl. Med. 40:883-888 (1999) DITWDQLWDLMK  99Koivunen et al., J. Nucl. Med. 40:883-888 (1999) CWDD(G/L)WLC 100Koivunen et al., J. Nucl. Med. 40:883-888 (1999) EWCEYLGGYLRCY 101Koivunen et al., J. Nucl. Med. 40:883-888 (1999) A YXCXXGPXTVVXCX 102Koivunen et al., J. Nucl. Med. 40:883-888 (1999) P IEGPTLRQWLAARA 103Koivunen et al., J. Nucl. Med. 40:883-888 (1999) LWXX(Y/W/F/H) 104Koivunen et al., J. Nucl. Med. 40:883-888 (1999) XFXXYLW 105Koivunen et al., J. Nucl. Med. 40:883-888 (1999) RWGLCD 1060Koivunen et al., J. Nucl. Med. 40:883-888 (1999) MSRPACPPNDKYE 107Koivunen et al., J. Nucl. Med. 40:883-888 (1999) CLRSGRGC 108Koivunen et al., J. Nucl. Med. 40:883-888 (1999) CHWMFSPWC 109Koivunen et al., J. Nucl. Med. 40:883-888 (1999) WXXF 110Koivunen et al., J. Nucl. Med. 40:883-888 (1999) CSSRLDAC 111Koivunen et al., J. Nucl. Med. 40:883-888 (1999) CLPVASC 112Koivunen et al., J. Nucl. Med. 40:883-888 (1999) CGFECVRQCPERC 113Koivunen et al., J. Nucl. Med. 40:883-888 (1999) CVALCREACGEGC 114Koivunen et al., J. Nucl. Med. 40:883-888 (1999) SWCEPGWCR 115Koivunen et al., J. Nucl. Med. 40:883-888 (1999) YSGWGW 116Koivunen et al., J. Nucl. Med. 40:883-888 (1999) GLSGGRS 117Koivunen et al., J. Nucl. Med. 40:883-888 (1999) LMLPRAD 118Koivunen et al., J. Nucl. Med. 40:883-888 (1999) CSCFRDVCC 119Koivunen et al., J. Nucl. Med. 40:883-888 (1999) CRDVVSVIC 120Koivunen et al., J. Nucl. Med. 40:883-888 (1999) CNGRC 121Koivunen et al., J. Nucl. Med. 40:883-888 (1999) MARSGL 122Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) MARAKE 123Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) MSRTMS 124Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) KCCYSL 125Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) MYWGDSHWLQYW 126Newton & Deutscher, Phage Peptide Display in YEHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) MQLPLAT 127Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) EWLS 128Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) SNEW 129Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) TNYL 130Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) WIFPWIQL 131Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) WDLAWMFRLPVG 132Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CTVALPGGYVRVC 133Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CVPELGHEC 134Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CGRRAGGSC 135Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CVAYCIEHHCWTC 136Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CVFAHNYDYLVC 137Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CVFTSNYAFC 138Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) VHSPNKK 139Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CDCRGDCFC 140Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CRGDGWC 141Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) XRGCDX 142Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) PXX(S/T) 143Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CTTHWGFTLC 144Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) SGKGPRQITAL 145Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) A(A/Q)(N/A)(L/Y)(T/V/ 146Newton & Deutscher, Phage Peptide Display in M/R)(R/K)Handbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) VYMSPF 147Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) MQLPLAT 148Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) ATWLPPR 149Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) HTMYYHHYQHHL 150Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) SEVGCRAGPLQWL 151Newton & Deutscher, Phage Peptide Display in CEKYFGHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CGLLPVGRPDRNV 152Newton & Deutscher, Phage Peptide Display in WRWLCHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CKGQCDRFKGLPW 153Newton & Deutscher, Phage Peptide Display in ECHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) SGRSA 154Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) WGFP 155Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) LVVXXAr 156Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) XFXXYLW 157Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) AEPMPHSLNFSQYL 158Newton & Deutscher, Phage Peptide Display in WYTHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) WAY(W/F)SP 159Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) IELLQAR 160Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) DITWDQLWDLMK 161Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) AYTKCSRQWRTCM 162Newton & Deutscher, Phage Peptide Display in TTHHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) PQNSKIPGPTFLDP 163Newton & Deutscher, Phage Peptide Display in HHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) SMEPALPDWWWK 164Newton & Deutscher, Phage Peptide Display in MFKHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) ANTPCGPYTHDCP 165Newton & Deutscher, Phage Peptide Display in VKRHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) TACHQHVRMVRP 166Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) VPWMEPAYQRFL 167Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) DPRATPGS 168Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) FRPNRAQDYNTN 169Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CTKNSYLMC 170Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) C(R/Q)L/RT(G/N)XX 171Newton & Deutscher, Phage Peptide Display in G(A/V)GCHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) CPIEDRPMC 172Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) HEWSYLAPYPWF 173Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) MCPKHPLGC 174Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) RMWPSSTVNLSAG 175Newton & Deutscher, Phage Peptide Display in RRHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) SAKTAVSQRVWLP 176Newton & Deutscher, Phage Peptide Display in SHRGGEPHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) KSREHVNNSACPS 177Newton & Deutscher, Phage Peptide Display in KRITAALHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) EGFR 178Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) AGLGVR 179Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) GTRQGHTMRLGVS 180Newton & Deutscher, Phage Peptide Display in DGHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) IAGLATPGWSHWLA 181Newton & Deutscher, Phage Peptide Display in LHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) SMSIARL 182Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) HTFEPGV 183Newton & Deutscher, Phage Peptide Display inHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) NTSLKRISNKR1RR 184Newton & Deutscher, Phage Peptide Display in KHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008) LRIKRKRRKRKKTR 185Newton & Deutscher, Phage Peptide Display in KHandbook of Experimental Pharmacology, pages145-163, Springer-Verlag, Berlin (2008)

As yet a further embodiment, the targeting sequence may be a peptidethat can be used for chemical coupling (e.g., can comprise arginineand/or lysine residues that can be chemically coupled through their Rgroups) to another molecule that targets entry into a cell.

As another embodiment, the AAV capsid protein or virus capsid of thedisclosure can comprise a mutation as described in WO 2006/066066. Forexample, the capsid protein can comprise a selective amino acidsubstitution at amino acid position 263, 705, 708 and/or 716 of thenative AAV2 capsid protein or a corresponding change(s) in a capsidprotein from another AAV serotype.

Additionally, or alternatively, in representative embodiments, thecapsid protein, virus capsid or vector comprises a selective amino acidinsertion directly following amino acid position 264 of the AAV2 capsidprotein or a corresponding change in the capsid protein from other AAV.By “directly following amino acid position X” it is intended that theinsertion immediately follows the indicated amino acid position (forexample, “following amino acid position 264” indicates a point insertionat position 265 or a larger insertion, e.g., from positions 265 to 268,etc.).

Furthermore, in representative embodiments, the capsid protein, viruscapsid or vector of this disclosure can comprise amino acidmodifications such as described in PCT Publication No. WO 2010/093784(e.g., 2i8) and/or in PCT Publication No. WO 2014/144229 (e.g., dualglycan).

In some embodiments of this disclosure, the capsid protein, virus capsidor vector of this disclosure can have equivalent or enhancedtransduction efficiency relative to the transduction efficiency of theAAV serotype from which the capsid protein, virus capsid or vector ofthis disclosure originated. In some embodiments of this disclosure, thecapsid protein, virus capsid or vector of this disclosure can havereduced transduction efficiency relative to the transduction efficiencyof the AAV serotype from which the capsid protein, virus capsid orvector of this disclosure originated. In some embodiments of thisdisclosure, the capsid protein, virus capsid or vector of thisdisclosure can have equivalent or enhanced tropism relative to thetropism of the AAV serotype from which the capsid protein, virus capsidor vector of this disclosure originated. In some embodiments of thisdisclosure, the capsid protein, virus capsid or vector of thisdisclosure can have an altered or different tropism relative to thetropism of the AAV serotype from which the capsid protein, virus capsidor vector of this disclosure originated. In some embodiments of thisdisclosure, the capsid protein, virus capsid or vector of thisdisclosure can have or be engineered to have tropism for brain tissue.In some embodiments of this disclosure, the capsid protein, virus capsidor vector of this disclosure can have or be engineered to have tropismfor liver tissue.

The foregoing embodiments can be used to deliver a heterologous nucleicacid to a cell or subject as described herein. For example, the modifiedvector can be used to treat a lysosomal storage disorder such as amucopolysaccharidosis disorder (e.g., Sly syndrome [β-glucuronidase],Hurler Syndrome [alpha-L-iduronidase], Scheie Syndrome[alpha-L-iduronidase], Hurler-Scheie Syndrome [alpha-L-iduronidase],Hunter's Syndrome [iduronate sulfatase], Sanfilippo Syndrome A [heparansulfamidase], B [N-acetylglucosaminidase], C[acetyl-CoA:alpha-glucosaminide acetyltransferase], D[N-acetylglucosamine 6-sulfatase], Morquio Syndrome A[galactose-6-sulfate sulfatase], B [β-galactosidase], Maroteaux-LamySyndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease(a-galactosidase), Gaucher's disease (glucocerebrosidase), or a glycogenstorage disorder (e.g., Pompe disease; lysosomal acid alpha-glucosidase)as described herein.

Those skilled in the art will appreciate that for some AAV capsidproteins the corresponding modification will be an insertion and/or asubstitution, depending on whether the corresponding amino acidpositions are partially or completely present in the virus or,alternatively, are completely absent.

The disclosure also encompasses virus vectors comprising the modifiedcapsid proteins and capsids of the disclosure. In particularembodiments, the virus vector is a parvovirus vector (e.g., comprising aparvovirus capsid and/or vector genome), for example, an AAV vector(e.g., comprising an AAV capsid and/or vector genome). In representativeembodiments, the virus vector comprises a modified AAV capsid comprisinga modified capsid subunit of the disclosure and a vector genome.

For example, in representative embodiments, the virus vector comprises:(a) a modified virus capsid (e.g., a modified AAV capsid) comprising amodified capsid protein of the disclosure; and (b) a nucleic acidcomprising a terminal repeat sequence (e.g., an AAV TR), wherein thenucleic acid comprising the terminal repeat sequence is encapsidated bythe modified virus capsid. The nucleic acid can optionally comprise twoterminal repeats (e.g., two AAV TRs).

In representative embodiments, the virus vector is a recombinant virusvector comprising a heterologous nucleic acid encoding a polypeptide orfunctional RNA of interest. Recombinant virus vectors are described inmore detail below.

In particular embodiments, the virus vectors of the disclosure (i) havereduced transduction of liver as compared with the level of transductionby a virus vector without the modified capsid protein; (ii) exhibitenhanced systemic transduction by the virus vector in an animal subjectas compared with the level observed by a virus vector without themodified capsid protein; (iii) demonstrate enhanced movement acrossendothelial cells as compared with the level of movement by a virusvector without the modified capsid protein, and/or (iv) exhibit aselective enhancement in transduction of an ophthalmic tissue (e.g.,retinal pigment epithelium), (v) exhibit a selective enhancement intransduction of liver tissue, and/or (vi) reduced transduction of braintissues (e.g., neurons) as compared with the level of transduction by avirus vector without the modified capsid protein.

It will be understood by those skilled in the art that the modifiedcapsid proteins, virus capsids and virus vectors of the disclosureexclude those capsid proteins, capsids and virus vectors that have theindicated amino acids at the specified positions in their native state(i.e., are not mutants).

Methods of Producing Virus Vectors

The present disclosure further provides methods of producing the virusvectors described herein. Thus, in one embodiment, the presentdisclosure provides a method of producing an AAV vector that hasenhanced tropism to a target tissue of interest (e.g. an ophthalmictissue), comprising: a) identifying amino acid residues that form athree dimensional surface-exposed loop on an AAV capsid protein; b)generating a library of AAV capsid proteins comprising amino acidsubstitutions of the amino acid residues identified in (a); c) producingAAV particles comprising capsid proteins from the library of AAV capsidproteins of (b); d) contacting the AAV particles of (c) with cells underconditions whereby infection and replication can occur; e) selecting AAVparticles that can complete at least one infectious cycle and replicateto titers similar to control AAV particles: 1) contacting the AAVparticles selected in (e) with cells of a target tissue of interestunder conditions whereby infection and replication can occur; and g)selecting AAV particles that transduce the target tissue of interest.Non-limiting examples of methods for identifying surface-exposed loopamino acid residues include peptide epitope mapping and/or cryo-electronmicroscopy.

Resolution and identification of the surface-exposed loop residueswithin the three dimensional surface-exposed loop allows for theirsubsequent modification through random, rational and/or degeneratemutagenesis to generate AAV capsids with desirable transduction patternsthat can be identified through further selection and/or screening.

Thus, in a further embodiment, the present disclosure provides a methodof producing an AAV vector that has an enhanced transduction profilewith respect to a target tissue of interest, comprising: a) identifyingamino acid residues that form a three dimensional surface-exposed loopon an AAV capsid protein; b) generating AAV capsid proteins comprisingamino acid substitutions of the surface-exposed loop amino acid residuesidentified in (a) by random, rational and/or degenerate mutagenesis; c)producing AAV particles comprising capsid proteins from the AAV capsidproteins of (b); d) contacting the AAV particles of (c) with cells underconditions whereby infection and replication can occur; e) selecting AAVparticles that can complete at least one infectious cycle and replicateto titers similar to control AAV particles; f) contacting the AAVparticles selected in (e) with cells of a target tissue of interestunder conditions whereby infection and replication can occur; and g)selecting AAV particles that transduce the target tissue of interest.

Nonlimiting examples of methods for identifying surface-exposed loopamino acid residues include peptide epitope mapping and/or cryo-electronmicroscopy. Methods of generating AAV capsid proteins comprising aminoacid substitutions of surface loop amino acid residues by random,rational and/or degenerate mutagenesis are known in the art.

This comprehensive approach presents a platform technology that can beapplied to modifying any AAV capsid. Application of this platformtechnology yields AAV variants derived from the original AAV capsidtemplate that have desirable transduction efficiency. As one advantageand benefit, application of this technology will expand the cohort ofpatients eligible for gene therapy with AAV vectors.

In one embodiment, the present disclosure provides a method of producinga virus vector, the method comprising providing to a cell: (a) a nucleicacid template comprising at least one TR sequence (e.g., AAV TRsequence), and (b) AAV sequences sufficient for replication of thenucleic acid template and encapsidation into AAV capsids (e.g., AAV repsequences and AAV cap sequences encoding the AAV capsids of thedisclosure). Optionally, the nucleic acid template further comprises atleast one heterologous nucleic acid sequence. In particular embodiments,the nucleic acid template comprises two AAV ITR sequences, which arelocated 5′ and 3′ to the heterologous nucleic acid sequence (ifpresent), although they need not be directly contiguous thereto.

The nucleic acid template and AAV rep and cap sequences are providedunder conditions such that virus vector comprising the nucleic acidtemplate packaged within the AAV capsid is produced in the cell. Themethod can further comprise the step of collecting the virus vector fromthe cell. The virus vector can be collected from the medium and/or bylysing the cells.

The cell can be a cell that is permissive for AAV viral replication. Anysuitable cell known in the art may be employed. In particularembodiments, the cell is a mammalian cell. As another option, the cellcan be a trans-complementing packaging cell line that provides functionsdeleted from a replication-defective helper virus, e.g., 293 cells orother E1a trans-complementing cells.

The AAV replication and capsid sequences may be provided by any methodknown in the art. Current protocols typically express the AAV rep/capgenes on a single plasmid. The AAV replication and packaging sequencesneed not be provided together, although it may be convenient to do so.The AAV rep and/or cap sequences may be provided by any viral ornon-viral vector. For example, the rep/cap sequences may be provided bya hybrid adenovirus or herpesvirus vector (e.g., inserted into the Elaor E3 regions of a deleted adenovirus vector). EBV vectors may also beemployed to express the AAV cap and rep genes. One advantage of thismethod is that EBV vectors are episomal, yet will maintain a high copynumber throughout successive cell divisions (i.e., are stably integratedinto the cell as extra-chromosomal elements, designated as an “EBV basednuclear episome,” see Margolski, (1992) Curr. Top. Microbiol. Immun.158:67).

As a further alternative, the rep/cap sequences may be stablyincorporated into a cell.

Typically the AAV rep/cap sequences will not be flanked by the TRs, toprevent rescue and/or packaging of these sequences.

The nucleic acid template can be provided to the cell using any methodknown in the art. For example, the template can be supplied by anon-viral (e.g., plasmid) or viral vector. In particular embodiments,the nucleic acid template is supplied by a herpesvirus or adenovirusvector (e.g., inserted into the Ela or E3 regions of a deletedadenovirus). As another illustration, Palombo et al., (1998) J. Virology72:5025, describes a baculovirus vector carrying a reporter gene flankedby the AAV TRs. EBV vectors may also be employed to deliver thetemplate, as described above with respect to the rep/cap genes.

In another representative embodiment, the nucleic acid template isprovided by a replicating rAAV virus. In still other embodiments, an AAVprovirus comprising the nucleic acid template is stably integrated intothe chromosome of the cell.

To enhance virus titers, helper virus functions (e.g., adenovirus orherpesvirus) that promote a productive AAV infection can be provided tothe cell. Helper virus sequences necessary for AAV replication are knownin the art. Typically, these sequences will be provided by a helperadenovirus or herpesvirus vector. Alternatively, the adenovirus orherpesvirus sequences can be provided by another non-viral or viralvector, e.g., as a noninfectious adenovirus miniplasmid that carries allof the helper genes that promote efficient AAV production as describedby Ferrari et al., (1997) Nature Med. 3: 1295, and U.S. Pat. Nos.6,040,183 and 6,093,570.

Further, the helper virus functions may be provided by a packaging cellwith the helper sequences embedded in the chromosome or maintained as astable extrachromosomal element. Generally, the helper virus sequencescannot be packaged into AAV virions, e.g., are not flanked by TRs.

Those skilled in the art will appreciate that it may be advantageous toprovide the AAV replication and capsid sequences and the helper virussequences (e.g., adenovirus sequences) on a single helper construct.This helper construct may be a non-viral or viral construct. As onenonlimiting illustration, the helper construct can be a hybridadenovirus or hybrid herpesvirus comprising the AAV rep/cap genes.

In one particular embodiment, the AAV rep/cap sequences and theadenovirus helper sequences are supplied by a single adenovirus helpervector. This vector further can further comprise the nucleic acidtemplate. The AAV rep/cap sequences and/or the rAAV template can beinserted into a deleted region (e.g., the Ela or E3 regions) of theadenovirus.

In a further embodiment, the AAV rep/cap sequences and the adenovirushelper sequences are supplied by a single adenovirus helper vector.According to this embodiment, the rAAV template can be provided as aplasmid template.

In another illustrative embodiment, the AAV rep/cap sequences andadenovirus helper sequences are provided by a single adenovirus helpervector, and the rAAV template is integrated into the cell as a provirus.Alternatively, the rAAV template is provided by an EBV vector that ismaintained within the cell as an extrachromosomal element (e.g., as anEBV based nuclear episome).

In a further exemplary embodiment, the AAV rep/cap sequences andadenovirus helper sequences are provided by a single adenovirus helper.The rAAV template can be provided as a separate replicating viralvector. For example, the rAAV template can be provided by a rAAVparticle or a second recombinant adenovirus particle.

According to the foregoing methods, the hybrid adenovirus vectortypically comprises the adenovirus 5′ and 3′ cis sequences sufficientfor adenovirus replication and packaging (i.e., the adenovirus terminalrepeats and PAC sequence). The AAV rep/cap sequences and, if present,the rAAV template are embedded in the adenovirus backbone and areflanked by the 5′ and 3′ cis sequences, so that these sequences may bepackaged into adenovirus capsids. As described above, the adenovirushelper sequences and the AAV rep/cap sequences are generally not flankedby TRs so that these sequences are not packaged into the AAV virions.

Zhang et al., ((2001) Gene Ther. 18:704-12) describe a chimeric helpercomprising both adenovirus and the AAV rep and cap genes.

Herpesvirus may also be used as a helper virus in AAV packaging methods.Hybrid herpesviruses encoding the AAV Rep protein(s) may advantageouslyfacilitate scalable AAV vector production schemes. A hybrid herpessimplex virus type I (HSV-1) vector expressing the AAV-2 rep and capgenes has been described (Conway et al., (1999) Gene Therapy 6:986 andWO 00/17377.

As a further alternative, the virus vectors of the disclosure can beproduced in insect cells using baculovirus vectors to deliver therep/cap genes and rAAV template as described, for example, by Urabe etal., (2002) Human Gene Therapy 13: 1935-43.

AAV vector stocks free of contaminating helper virus may be obtained byany method known in the art. For example, AAV and helper virus may bereadily differentiated based on size. AAV may also be separated awayfrom helper virus based on affinity for a heparin substrate (Zolotukhinet al. (1999) Gene Therapy 6:973). Deleted replication-defective helperviruses can be used so that any contaminating helper virus is notreplication competent. As a further alternative, an adenovirus helperlacking late gene expression may be employed, as only adenovirus earlygene expression is required to mediate packaging of AAV virus.Adenovirus mutants defective for late gene expression are known in theart (e.g., ts100K and ts149 adenovirus mutants).

Recombinant Virus Vectors

The virus vectors of the present disclosure are useful for the deliveryof nucleic acids to cells in vitro, ex vivo, and in vivo. In particular,the virus vectors can be advantageously employed to deliver or transfernucleic acids to animal, including mammalian, cells. Thus, in someembodiments, a nucleic acid (“cargo nucleic acid”) may be encapsidatedby a capsid protein of the disclosure.

In some embodiments, the disclosure provides an AAV vector comprising arecombinant capsid protein with at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity, with any one of SEQ ID NO: 11-12, 23-49, or 195-254. In someembodiments, an AAV vector comprising a recombinant capsid protein withat least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity, with any one of SEQ ID NO: 32, 35,37, or 40. In some embodiments, an AAV viral vector comprises arecombinant capsid protein with at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity, with any one of SEQ ID NO: 11-12, 23-49, or 195-254 andfurther comprises a cargo nucleic acid encapsidated by the capsidprotein. In some embodiments, an AAV viral vector comprises arecombinant capsid protein with at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity, with any one of SEQ ID NO: 32, 35, 37, or 40 and furthercomprises a cargo nucleic acid encapsidated by the capsid protein. Insome embodiments, the AAV vectors specifically target and infect atissue of the eye (such as the RPE or the retina). In some embodiments,the AAV vectors display enhanced transduction to the eye (e.g., the RPEor the retina) compared to a parental AAV capsid.

The cargo nucleic acid sequence delivered in the virus vectors of thepresent disclosure may be any heterologous nucleic acid sequence(s) ofinterest. Nucleic acids of interest include nucleic acids encodingpolypeptides, including therapeutic (e.g., for medical or veterinaryuses) or immunogenic (e.g., for vaccines) polypeptides or RNAs.

Therapeutic polypeptides include, but are not limited to, cysticfibrosis transmembrane regulator protein (CFTR), dystrophin (includingmini- and micro-dystrophins, see, e.g., Vincent et al, (1993) NatureGenetics 5: 130; U.S. Patent Publication No. 2003/017131; Internationalpublication WO/2008/088895, Wang et al., Proc. Natl. Acad. Sci. USA 97:1 3714-13719 (2000); and Gregorevic et al., Mol. Ther. 16:657-64(2008)), myostatin propeptide, follistatin, activin type 11 solublereceptor, IGF-1, apolipoproteins such as apoA (apoA1, apoA2, apoA4,apoA-V), apoB (apoB100, ApoB48), apoC (apoCI, apoClI, apoCIII, apoCIV),apoD, apoE, apoH, apoL, apo(a), anti-inflammatory polypeptides such asthe lkappa B dominant mutant, amyloid beta, tau, sarcospan, utrophin(Tinsley et al, (1996) Nature 384:349), mini-utrophin, clotting factors(e.g., Factor VIII, Factor IX, Factor X, etc.), erythropoietin,angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxidedismutase, leptin, the LDL receptor, lipoprotein lipase, progranulin,ornithine transcarbamylase, β-globin, α-globin, spectrin,alpha-1-antitrypsin, adenosine deaminase, hypoxanthine guaninephosphoribosyl transferase, β-glucocerebrosidase, battenin,sphingomyelinase, lysosomal hexosaminidase A, branched-chain keto aciddehydrogenase, frataxin, RP65 protein, cytokines (e.g.,alpha-interferon, beta-interferon, gamma-interferon, interleukin-2,interleukin-4, alpha synuclein, parkin, granulocyte-macrophage colonystimulating factor, lymphotoxin, and the like), peptide growth factors,neurotrophic factors and hormones (e.g., somatotropin, insulin,insulin-like growth factors 1 and 2, platelet derived growth factor,epidermal growth factor, fibroblast growth factor, nerve growth factor,neurotrophic factor-3 and -4, brain-derived neurotrophic factor, bonemorphogenic proteins [including RANKL and VEGF], glial derived growthfactor, transforming growth factor-α and -β, and the like), huntingtin,lysosomal acid alpha-glucosidase, iduronate-2-sulfatase,N-sulfoglucosamine sulfohydrolase, alpha-galactosidase A, receptors(e.g., the tumor necrosis growth factor soluble receptor), S100A1,ubiquitin protein ligase E3, parvalbumin, adenylyl cyclase type 6, amolecule that modulates calcium handling (e.g., SERCA2A, Inhibitor 1 ofPP1 and fragments thereof [e.g., WO 2006/029319 and WO 2007/100465]), amolecule that effects G-protein coupled receptor kinase type 2 knockdownsuch as a truncated constitutively active bARKct, anti-inflammatoryfactors such as IRAP, anti-myostatin proteins, aspartoacylase,monoclonal antibodies (including single chain monoclonal antibodies; anexemplary Mab is the Herceptin® Mab), neuropeptides and fragmentsthereof (e.g., galanin, Neuropeptide Y (see, U.S. Pat. No. 7,071,172),angiogenesis inhibitors such as Vasohibins and other VEGF inhibitors(e.g., Vasohibin 2 [see, WO JP2006/073052]). Other illustrativeheterologous nucleic acid sequences encode suicide gene products (e.g.,thymidine kinase, cytosine deaminase, diphtheria toxin, and tumornecrosis factor), proteins that enhance or inhibit transcription of hostfactors (e.g., nuclease-dead Cas9 linked to a transcription enhancer orinhibitor element, zinc-finger proteins linked to a transcriptionenhancer or inhibitor element, transcription activator-like (TAL)effectors linked to a transcription enhancer or inhibitor element),proteins conferring resistance to a drug used in cancer therapy, tumorsuppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, FAS-ligand, RS1,opsin, TKR-beta, C3, CFH, and any other polypeptide that has atherapeutic effect in a subject in need thereof. In some embodiments, anAAV vector may be used to deliver a complement protein such as C21a,C4b, C3a, C3b, C5a, C5b, C6, C7, C8, or C9. AAV vectors can also be usedto deliver monoclonal antibodies and antibody fragments, for example, anantibody or antibody fragment directed against myostatin (see, e.g.,Fang et al., Nature Biotechnology 23:584-590 (2005)). Heterologousnucleic acid sequences encoding polypeptides include those encodingreporter polypeptides (e.g., an enzyme). Reporter polypeptides are knownin the art and include, but are not limited to, Green FluorescentProtein, 3-galactosidase, alkaline phosphatase, luciferase, andchloramphenicol acetyltransferase gene.

Optionally, the heterologous nucleic acid encodes a secreted polypeptide(e.g., a polypeptide that is a secreted polypeptide in its native stateor that has been engineered to be secreted, for example, by operableassociation with a secretory signal sequence as is known in the art).

Alternatively, in particular embodiments of this disclosure, theheterologous nucleic acid may encode an antisense nucleic acid, aribozyme (e.g., as described in U.S. Pat. No. 5,877,022), RNAs thateffect spliceosome-mediated/ram-splicing (see, Puttaraju et al, (1999)Nature Biotech. 17:246; U.S. Pat. Nos. 6,013,487; 6,083,702),interfering RNAs (RNAi) including siRNA, shRNA or miRNA that mediategene silencing (see, Sharp et al, (2000) Science 287:2431), and othernon-translated RNAs, such as “guide” RNAs (Gorman et al., (1998) Proc.Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.),and the like. Exemplary untranslated RNAs include RNAi against amultiple drug resistance (MDR) gene product (e.g., to treat and/orprevent tumors and/or for administration to the heart to prevent damageby chemotherapy), RNAi against myostatin (e.g., for Duchenne musculardystrophy), RNAi against VEGF (e.g., to treat and/or prevent tumors),RNAi against phospholamban (e.g., to treat cardiovascular disease, see,e.g., Andino et al., J. Gene Med. 10: 132-142 (2008) and Li et al., ActaPharmacol Sin. 26:51-55 (2005)); phospholamban inhibitory ordominant-negative molecules such as phospholamban S 16E (e.g., to treatcardiovascular disease, see, e.g., Hoshijima et al. Nat. Med. 8:864-871(2002)), RNAi to adenosine kinase (e.g., for epilepsy), and RNAidirected against pathogenic organisms and viruses (e.g., hepatitis Band/or C virus, human immunodeficiency virus, CMV, herpes simplex virus,human papilloma virus, etc.).

Further, a nucleic acid sequence that directs alternative splicing canbe delivered. To illustrate, an antisense sequence (or other inhibitorysequence) complementary to the 5′ and/or 3′ splice site of dystrophinexon 51 can be delivered in conjunction with a U1 or U7 small nuclear(sn) RNA promoter to induce skipping of this exon. For example, a DNAsequence comprising a U1 or U7 snRNA promoter located 5′ to theantisense/inhibitory sequence(s) can be packaged and delivered in amodified capsid of the disclosure.

In some embodiments, a nucleic acid sequence that directs gene editingcan be delivered. For example, the nucleic acid may encode a guide RNA.In some embodiments, the guide RNA is a single guide RNA (sgRNA)comprising a crRNA sequence and a tracrRNA sequence. In someembodiments, the nucleic acid may encode a nuclease. In someembodiments, the nuclease is a zinc-finger nuclease, a homingendonuclease, a TALEN (transcription activator-like effector nuclease),a NgAgo (agronaute endonuclease), a SGN (structure-guided endonuclease),a RGN (RNA-guided nuclease), or modified or truncated variants thereof.In some embodiments, the RNA-guided nuclease is a Cas9 nuclease, aCas12(a) nuclease (Cpf1), a Cas12b nuclease, a Cas12c nuclease, aTrpB-like nuclease, a Cas13a nuclease (C2c2), a Cas13b nuclease, a Cas14nuclease, or modified or truncated variants thereof. In someembodiments, the Cas9 nuclease is isolated or derived from S. pyogenesor S. aureus.

In some embodiments, a nucleic acid sequence that directs gene knockdowncan be delivered. For example, the nucleic acid sequence may encode asiRNA, an shRNA, a microRNA, or an antisense nucleic acid. The virusvector may also comprise a heterologous nucleic acid that shareshomology with and recombines with a locus on a host chromosome. Thisapproach can be utilized, for example, to correct a genetic defect inthe host cell.

The virus vector may also comprise a heterologous nucleic acid thatshares homology with and recombines with a locus on a host chromosome.This approach can be utilized, for example, to correct a genetic defectin the host cell.

The present disclosure also provides virus vectors that express animmunogenic polypeptide, e.g., for vaccination. The nucleic acid mayencode any immunogen of interest known in the art including, but notlimited to, immunogens from human immunodeficiency virus (HIV), simianimmunodeficiency virus (SIV), influenza virus, HIV or SIV gag proteins,tumor antigens, cancer antigens, bacterial antigens, viral antigens, andthe like.

The use of parvoviruses as vaccine vectors is known in the art (see,e.g., Miyamura el al, (1994) Proc. Nat. Acad. Sci USA 91:8507; U.S. Pat.No. 5,916,563 to Young et al, U.S. Pat. No. 5,905,040 to Mazzara et al,U.S. Pat. Nos. 5,882,652, 5,863,541 to Samulski et al). The antigen maybe presented in the parvovirus capsid.

Alternatively, the antigen may be expressed from a heterologous nucleicacid introduced into a recombinant vector genome. Any immunogen ofinterest as described herein and/or as is known in the art can beprovided by the virus vector of the present disclosure.

An immunogenic polypeptide can be any polypeptide suitable for elicitingan immune response and/or protecting the subject against an infectionand/or disease, including, but not limited to, microbial, bacterial,protozoal, parasitic, fungal and/or viral infections and diseases. Forexample, the immunogenic polypeptide can be an orthomyxovirus immunogen(e.g., an influenza virus immunogen, such as the influenza virushemagglutinin (HA) surface protein or the influenza virus nucleoprotein,or an equine influenza virus immunogen) or a lentivirus immunogen (e.g.,an equine infectious anemia virus immunogen, a Simian ImmunodeficiencyVirus (SIV) immunogen, or a Human Immunodeficiency Virus (HIV)immunogen, such as the HIV or SIV envelope GP 160 protein, the HIV orSIV matrix/capsid proteins, and the HIV or SIV gag, pol and env genesproducts). The immunogenic polypeptide can also be an arenavirusimmunogen (e.g., Lassa fever virus immunogen, such as the Lassa fevervirus nucleocapsid protein and the Lassa fever envelope glycoprotein), apoxvirus immunogen (e.g., a vaccinia virus immunogen, such as thevaccinia LI or L8 gene products), a flavivirus immunogen (e.g., a yellowfever virus immunogen or a Japanese encephalitis virus immunogen), afilovirus immunogen (e.g., an Ebola virus immunogen, or a Marburg virusimmunogen, such as NP and GP gene products), a bunyavirus immunogen(e.g., RVFV, CCHF, and/or SFS virus immunogens), or a coronavirusimmunogen (e.g., an infectious human coronavirus immunogen, such as thehuman coronavirus envelope glycoprotein, or a porcine transmissiblegastroenteritis virus immunogen, or an avian infectious bronchitis virusimmunogen). The immunogenic polypeptide can further be a polioimmunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogens), a mumpsimmunogen, a measles immunogen, a rubella immunogen, a diphtheria toxinor other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g.,hepatitis A, hepatitis B, hepatitis C, etc.) immunogen, and/or any othervaccine immunogen now known in the art or later identified as animmunogen.

Alternatively, the immunogenic polypeptide can be any tumor or cancercell antigen. Optionally, the tumor or cancer antigen is expressed onthe surface of the cancer cell.

Exemplary cancer and tumor cell antigens are described in S. A.Rosenberg (Immunity 10:281 (1991)). Other illustrative cancer and tumorantigens include, but are not limited to: BRCA1 gene product, BRCA2 geneproduct, gp100, tyrosinase, GAGE-1/2, BALE, RAGE, LAGE, NY-ESO-1, CDK-4,β-catenin, MUM-1, Caspase-8, KIAA0205, HPVE, SART-1, FRAME, p15,melanoma tumor antigens (Kawakami et al., (1994) Proc. Natl. Acad. Sci.USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami etal., (1994) Cancer Res. 54:3124), MART-1, gp100, MAGE-1, MAGE-2, MAGE-3,CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al., (1993) J Exp. Med.178:489); HER-2/neu gene product (U.S. Pat. No. 4,968,603), CA 125,LK26, FB5 (endosialin), TAG 72, AFP, CA 19-9, NSE, DU-PAN-2, CA50,SPan-1, CA72-4, HCG, STN (sialyl Tn antigen), c-erbB-2 proteins, PSA,L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressorprotein (Levine, (1993) Ann. Rev. Biochem. 62:623); mucin antigens(International Patent Publication No. WO 90/05142); telomerases; nuclearmatrix proteins; prostatic acid phosphatase; papilloma virus antigens;and/or antigens now known or later discovered to be associated with thefollowing cancers: melanoma, adenocarcinoma, thymoma, lymphoma (e.g.,non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer, livercancer, colon cancer, leukemia, uterine cancer, breast cancer, prostatecancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer,pancreatic cancer, brain cancer and any other cancer or malignantcondition or metastasis thereof now known or later identified (see,e.g., Rosenberg, (1996) Ann. Rev. Med. 47:481-91).

As a further alternative, the heterologous nucleic acid can encode anypolypeptide that is desirably produced in a cell in vitro, ex vivo, orin vivo. For example, the virus vectors may be introduced into culturedcells and the expressed gene product isolated therefrom.

It will be understood by those skilled in the art that the heterologousnucleic acid(s) of interest can be operably associated with appropriatecontrol sequences. For example, the heterologous nucleic acid can beoperably associated with expression control elements, such astranscription/translation control signals, origins of replication,polyadenylation signals, internal ribosome entry sites (IRES),promoters, and/or enhancers, and the like.

Further, regulated expression of the heterologous nucleic acid(s) ofinterest can be achieved at the post-transcriptional level, e.g., byregulating selective splicing of different introns by the presence orabsence of an oligonucleotide, small molecule and/or other compound thatselectively blocks splicing activity at specific sites (e.g., asdescribed in WO 2006/119137).

Those skilled in the art will appreciate that a variety ofpromoter/enhancer elements can be used depending on the level andtissue-specific expression desired. The promoter/enhancer can beconstitutive or inducible, depending on the pattern of expressiondesired. The promoter/enhancer can be native or foreign and can be anatural or a synthetic sequence. By foreign, it is intended that thetranscriptional initiation region is not found in the wild-type hostinto which the transcriptional initiation region is introduced.

In particular embodiments, the promoter/enhancer elements can be nativeto the target cell or subject to be treated. In representativeembodiments, the promoters/enhancer element can be native to theheterologous nucleic acid sequence. The promoter/enhancer element isgenerally chosen so that it functions in the target cell(s) of interest.Further, in particular embodiments the promoter/enhancer element is amammalian promoter/enhancer element. The promoter/enhancer element maybe constitutive or inducible.

Inducible expression control elements are typically advantageous inthose applications in which it is desirable to provide regulation overexpression of the heterologous nucleic acid sequence(s). Induciblepromoters/enhancer elements for gene delivery can be tissue-specific or-preferred promoter/enhancer elements, and include muscle specific orpreferred (including cardiac, skeletal and/or smooth muscle specific orpreferred), neural tissue specific or preferred (includingbrain-specific or preferred), eye specific or preferred (includingretina-specific and cornea-specific), liver specific or preferred, bonemarrow specific or preferred, pancreatic specific or preferred, spleenspecific or preferred, and lung specific or preferred promoter/enhancerelements. Other inducible promoter/enhancer elements includehormone-inducible and metal-inducible elements. Exemplary induciblepromoters/enhancer elements include, but are not limited to, a Teton/off element, a RU486-inducible promoter, an ecdysone-induciblepromoter, a rapamycin-inducible promoter, and a metallothioneinpromoter.

In embodiments wherein the heterologous nucleic acid sequence(s) istranscribed and then translated in the target cells, specific initiationsignals are generally included for efficient translation of insertedprotein coding sequences. These exogenous translational controlsequences, which may include the ATG initiation codon and adjacentsequences, can be of a variety of origins, both natural and synthetic.

The virus vectors according to the present disclosure provide a meansfor delivering heterologous nucleic acids into a broad range of cells,including dividing and non-dividing cells. The virus vectors can beemployed to deliver a nucleic acid of interest to a cell in vitro, e.g.,to produce a polypeptide in vitro or for ex vivo gene therapy. The virusvectors are additionally useful in a method of delivering a nucleic acidto a subject in need thereof e.g., to express an immunogenic ortherapeutic polypeptide or a functional RNA. In this manner, thepolypeptide or functional RNA can be produced in vivo in the subject.The subject can be in need of the polypeptide because the subject has adeficiency of the polypeptide. Further, the method can be practicedbecause the production of the polypeptide or functional RNA in thesubject may impart some beneficial effect.

The virus vectors can also be used to produce a polypeptide of interestor functional RNA in cultured cells or in a subject (e.g., using thesubject as a bioreactor to produce the polypeptide or to observe theeffects of the functional RNA on the subject, for example, in connectionwith screening methods).

In general, the virus vectors of the present disclosure can be employedto deliver a heterologous nucleic acid encoding a polypeptide orfunctional RNA to treat and/or prevent any disease state for which it isbeneficial to deliver a therapeutic polypeptide or functional RNA.Illustrative disease states include, but are not limited to: cysticfibrosis (cystic fibrosis transmembrane regulator protein) and otherdiseases of the lung, hemophilia A (Factor VIII), hemophilia B (FactorIX), thalassemia (β-globin), anemia (erythropoietin) and other blooddisorders. Alzheimer's disease (GDF; neprilysin), multiple sclerosis(β-interferon), Parkinson's disease (glial-cell line derivedneurotrophic factor [GDNF]), Huntington's disease (RNAi to removerepeats), Canavan's disease, amyotrophic lateral sclerosis, epilepsy(galanin, neurotrophic factors), and other neurological disorders,cancer (endostatin, angiostatin, TRAIL, FAS-ligand, cytokines includinginterferons; RNAi including RNAi against VEGF or the multiple drugresistance gene product, mir-26a [e.g., for hepatocellular carcinoma]),diabetes mellitus (insulin), muscular dystrophies including Duchenne(dystrophin, mini-dystrophin, insulin-like growth factor I, asarcoglycan [e.g., α, β, γ], RNAi against myostatic myostatinpropeptide, follistatin, activin type II soluble receptor,anti-inflammatory polypeptides such as the lkappa B dominant mutant,sarcospan, utrophin, mini-utrophin, antisense or RNAi against splicejunctions in the dystrophin gene to induce exon skipping [see, e.g.,WO/2003/095647], antisense against U7 snRNAs to induce exon skipping[see, e.g., WO/2006/021724], and antibodies or antibody fragmentsagainst myostatin or myostatin propeptide) and Becker musculardystrophy, Myotonic dystrophy 1 or 2, facioscapulohumeral musculardystrophy (FSHD), Gaucher disease (glucocerebrosidase), Hurler's disease(a-L-iduronidase), adenosine deaminase deficiency (adenosine deaminase),glycogen storage diseases (e.g., Fabry disease [a-galactosidase] andPompe disease [lysosomal acid alpha-glucosidase]) and other metabolicdisorders, congenital emphysema (alpha-1-antitrypsin), Lesch-NyhanSyndrome (hypoxan thine guanine phosphoribosyl transferase),Niemann-Pick disease (sphingomyelinase), Tay-Sachs disease (lysosomalhexosaminidase A), frontotemporal dementia, Maple Syrup Urine Disease(branched-chain keto acid dehydrogenase), retinal degenerative diseases(and other diseases of the eye and retina; e.g., PDGF for maculardegeneration and/or vasohibin or other inhibitors of VEGF or otherangiogenesis inhibitors to treat/prevent retinal disorders, e.g., inType I diabetes), diseases of solid organs such as brain (includingParkinson's Disease [GDNF], astrocytomas [endostatin, angiostatin and/orRNAi against VEGF], glioblastomas [endostatin, angiostatin and/or RNAiagainst VEGF]), liver, kidney, heart including congestive heart failureor peripheral artery disease (PAD) (e.g., by delivering proteinphosphatase inhibitor I (1-1) and fragments thereof (e.g., 11C),serca2a, zinc finger proteins that regulate the phospholamban gene,Barkct, [32-adrenergic receptor, 2-adrenergic receptor kinase (BARK),phosphoinositide-3 kinase (PI3 kinase), S100A1, parvalbumin, adenylylcyclase type 6, a molecule that effects G-protein coupled receptorkinase type 2 knockdown such as a truncated constitutively activebARKct; calsarcin, RNAi against phospholamban; phospholamban inhibitoryor dominant-negative molecules such as phospholamban S16E, etc.),arthritis (insulin-like growth factors), joint disorders (insulin-likegrowth factor 1 and/or 2), intimal hyperplasia (e.g., by deliveringenos, inos), improve survival of heart transplants (superoxidedismutase), AIDS (soluble CD4), muscle wasting (insulin-like growthfactor 1), kidney deficiency (erythropoietin), anemia (erythropoietin),arthritis (anti-inflammatory factors such as I RAP and TNFa solublereceptor), hepatitis (a-interferon), LDL receptor deficiency (LDLreceptor), hyperammonemia (ornithine transcarbamylase), Krabbe's disease(galactocerebrosidase), Batten's disease, spinal cerebral ataxiasincluding SCA1, SCA2 and SCA3, phenylketonuria (phenylalaninehydroxylase), autoimmune diseases, and the like. The disclosure canfurther be used following organ transplantation to increase the successof the transplant and/or to reduce the negative side effects of organtransplantation or adjunct therapies (e.g., by administeringimmunosuppressant agents or inhibitory nucleic acids to block cytokineproduction). As another example, bone morphogenic proteins (includingBNP 2, 7, etc., RANKL and/or VEGF) can be administered with a boneallograft, for example, following a break or surgical removal in acancer patient.

In some embodiments, the virus vectors of the present disclosure can beemployed to deliver a heterologous nucleic acid encoding a polypeptideor functional RNA to treat and/or prevent a liver disease or disorder.The liver disease or disorder may be, for example, primary biliarycirrhosis, nonalcoholic fatty liver disease (NAFLD), non-alcoholicsteatohepatitis (NASH), autoimmune hepatitis, hepatitis B, hepatitis C,alcoholic liver disease, fibrosis, jaundice, primary sclerosingcholangitis (PSC), Budd-Chiari syndrome, hemochromatosis, Wilson'sdisease, alcoholic fibrosis, non-alcoholic fibrosis, liver steatosis,Gilbert's syndrome, biliary atresia, alpha-1-antitrypsin deficiency,alagille syndrome, progressive familial intrahepatic cholestasis,Hemophilia B, Hereditary Angioedema (HAE), Homozygous FamilialHypercholesterolemia (HoFH), Heterozygous Familial Hypercholesterolemia(HeFH), Von Gierke's Disease (GSD I), Hemophilia A, MethylmalonicAcidemia, Propionic Acidemia, Homocystinuria, Phenylketonuria (PKU),Tyrosinemia Type 1, Arginase 1 Deficiency, Argininosuccinate LyaseDeficiency, Carbamoyl-phosphate synthetase 1 deficiency, CitrullinemiaType 1, Citrin Deficiency, Crigler-Najjar Syndrome Type 1, Cystinosis,Fabry Disease, Glycogen Storage Disease 1b, LPL Deficiency,N-Acetylglutamate Synthetase Deficiency, Ornithine TranscarbamylaseDeficiency, Ornithine Translocase Deficiency, Primary Hyperoxaluria Type1, or ADA SCID.

The disclosure can also be used to produce induced pluripotent stemcells (iPS). For example, a virus vector of the disclosure can be usedto deliver stem cell associated nucleic acid(s) into a non-pluripotentcell, such as adult fibroblasts, skin cells, liver cells, renal cells,adipose cells, cardiac cells, neural cells, epithelial cells,endothelial cells, and the like.

Nucleic acids encoding factors associated with stem cells are known inthe art. Nonlimiting examples of such factors associated with stem cellsand pluripotency include Oct-3/4, the SOX family (e.g., SOX 1, SOX2,SOX3 and/or SOX 15), the KIf family (e.g., Klfl, KHZ Klf4 and/or Klf5),the Myc family (e.g., C-myc, L-myc and/or N-myc), NANOG and/or LIN28.

The disclosure can also be practiced to treat and/or prevent a metabolicdisorder such as diabetes (e.g., insulin), hemophilia (e.g., Factor IXor Factor VIII), a lysosomal storage disorder such as amucopolysaccharidosis disorder (e.g., Sly syndrome [β-glucuronidase]Hurler Syndrome [alpha-L-iduronidase], Scheie Syndrome[alpha-L-iduronidase], Hurler-Scheie Syndrome [alpha-L-iduronidase],Hunter's Syndrome [iduronate sulfatase], Sanfilippo Syndrome A [heparansulfamidase], B [N-acetylglucosaminidase], C[acetyl-CoA:alpha-glucosaminide acetyltransferase], D[N-acetylglucosamine 6-sulfatase], Morquio Syndrome A[galactoses-sulfate sulfatase], B [β-galactosidase], Maroteaux-LamySyndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease(alpha-galactosidase), Gaucher's disease (glucocerebrosidase), or aglycogen storage disorder (e.g., Pompe disease; lysosomal acidalpha-glucosidase).

Gene transfer has substantial use for understanding and providingtherapy for disease states. There are a number of inherited diseases inwhich defective genes are known and have been cloned. In general, theabove disease states fall into two classes: deficiency states, usuallyof enzymes, which are generally inherited in a recessive manner, andunbalanced states, which may involve regulatory or structural proteins,and which are typically inherited in a dominant manner. For deficiencystate diseases, gene transfer can be used to bring a normal gene intoaffected tissues for replacement therapy, as well as to create animalmodels for the disease using antisense mutations. For unbalanced diseasestates, gene transfer can be used to create a disease state in a modelsystem, which can then be used in efforts to counteract the diseasestate. Thus, virus vectors according to the present disclosure permitthe treatment and/or prevention of genetic diseases.

The virus vectors according to the present disclosure may also beemployed to provide a functional RNA to a cell in vitro or in vivo. Thefunctional RNA may be, for example, a non-coding RNA. In someembodiments, expression of the functional RNA in the cell can diminishexpression of a particular target protein by the cell. Accordingly,functional RNA can be administered to decrease expression of aparticular protein in a subject in need thereof. In some embodiments,expression of the functional RNA in the cell can increase expression ofa particular target protein by the cell. Accordingly, functional RNA canbe administered to increase expression of a particular protein in asubject in need thereof. In some embodiments, expression of thefunctional RNA can regulate splicing of a particular target RNA in acell. Accordingly, functional RNA can be administered to regulatesplicing a particular RNA in a subject in need thereof. In someembodiments, expression of the functional RNA in the cell can regulatethe function of a particular target protein by the cell. Accordingly,functional RNA can be administered to regulate the function of aparticular protein in a subject in need thereof. Functional RNA can alsobe administered to cells in vitro to regulate gene expression and/orcell physiology, e.g., to optimize cell or tissue culture systems or inscreening methods.

In addition, virus vectors according to the instant disclosure find usein diagnostic and screening methods, whereby a nucleic acid of interestis transiently or stably expressed in a cell culture system, oralternatively, a transgenic animal model.

The virus vectors of the present disclosure can also be used for variousnon-therapeutic purposes, including but not limited to use in protocolsto assess gene targeting, clearance, transcription, translation, etc.,as would be apparent to one skilled in the art. The virus vectors canalso be used for the purpose of evaluating safety (spread, toxicity,immunogenicity, etc.). Such data, for example, are considered by theUnited States Food and Drug Administration as part of the regulatoryapproval process prior to evaluation of clinical efficacy.

As a further aspect, the virus vectors of the present disclosure may beused to produce an immune response in a subject. According to thisembodiment, a virus vector comprising a heterologous nucleic acidsequence encoding an immunogenic polypeptide can be administered to asubject, and an active immune response is mounted by the subject againstthe immunogenic polypeptide. Immunogenic polypeptides are as describedhereinabove. In some embodiments, a protective immune response iselicited.

Alternatively, the virus vector may be administered to a cell ex vivoand the altered cell is administered to the subject. The virus vectorcomprising the heterologous nucleic acid is introduced into the cell,and the cell is administered to the subject, where the heterologousnucleic acid encoding the immunogen can be expressed and induce animmune response in the subject against the immunogen. In particularembodiments, the cell is an antigen-presenting cell (e.g., a dendriticcell).

An “active immune response” or “active immunity” is characterized by“participation of host tissues and cells after an encounter with theimmunogen. It involves differentiation and proliferation ofimmunocompetent cells in lymphoreticular tissues, which lead tosynthesis of antibody or the development of cell-mediated reactivity, orboth.” Herbert B. Herscowitz, Immunophysiology: Cell Function andCellular Interactions in Antibody Formation, in IMMUNOLOGY: BASICPROCESSES 117 (Joseph A. Bellanti ed., 1985). Alternatively stated, anactive immune response is mounted by the host after exposure to animmunogen by infection or by vaccination. Active immunity can becontrasted with passive immunity, which is acquired through the transferof preformed substances (antibody, transfer factor, thymic graft,interleukin-2) from an actively immunized host to a non-immune host.

A “protective” immune response or “protective” immunity as used hereinindicates that the immune response confers some benefit to the subjectin that it prevents or reduces the incidence of disease. Alternatively,a protective immune response or protective immunity may be useful in thetreatment and/or prevention of disease, in particular cancer or tumors(e.g., by preventing cancer or tumor formation, by causing regression ofa cancer or tumor and/or by preventing metastasis and/or by preventinggrowth of metastatic nodules). The protective effects may be complete orpartial, as long as the benefits of the treatment outweigh anydisadvantages thereof.

In particular embodiments, the virus vector or cell comprising theheterologous nucleic acid can be administered in an immunogenicallyeffective amount, as described below.

The virus vectors of the present disclosure can also be administered forcancer immunotherapy by administration of a virus vector expressing oneor more cancer cell antigens (or an immunologically similar molecule) orany other immunogen that produces an immune response against a cancercell. To illustrate, an immune response can be produced against a cancercell antigen in a subject by administering a virus vector comprising aheterologous nucleic acid encoding the cancer cell antigen, for exampleto treat a patient with cancer and/or to prevent cancer from developingin the subject. The virus vector may be administered to a subject invivo or by using ex vivo methods, as described herein.

Alternatively, the cancer antigen can be expressed as part of the viruscapsid or be otherwise associated with the virus capsid (e.g., asdescribed above).

As another alternative, any other therapeutic nucleic acid (e.g., RNAi)or polypeptide (e.g., cytokine) known in the art can be administered totreat and/or prevent cancer.

As used herein, the term “cancer” encompasses tumor-forming cancers.Likewise, the term “cancerous tissue” encompasses tumors. A “cancer cellantigen” encompasses tumor antigens.

The term “cancer” has its understood meaning in the art, for example, anuncontrolled growth of tissue that has the potential to spread todistant sites of the body (i.e., metastasize). Exemplary cancersinclude, but are not limited to melanoma, adenocarcinoma, thymoma,lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma,lung cancer, liver cancer, colon cancer, leukemia, uterine cancer,breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladdercancer, kidney cancer, pancreatic cancer, brain cancer and any othercancer or malignant condition now known or later identified. Inrepresentative embodiments, the disclosure provides a method of treatingand/or preventing tumor-forming cancers.

The term “tumor” is also understood in the art, for example, as anabnormal mass of undifferentiated cells within a multicellular organism.Tumors can be malignant or benign. In representative embodiments, themethods disclosed herein are used to prevent and treat malignant tumors.

By the terms “treating cancer,” “treatment of cancer” and equivalentterms it is intended that the severity of the cancer is reduced or atleast partially eliminated and/or the progression of the disease isslowed and/or controlled and/or the disease is stabilized. In particularembodiments, these terms indicate that metastasis of the cancer isprevented or reduced or at least partially eliminated and/or that growthof metastatic nodules is prevented or reduced or at least partiallyeliminated.

By the terms “prevention of cancer” or “preventing cancer” andequivalent terms it is intended that the methods at least partiallyeliminate or reduce and/or delay the incidence and/or severity of theonset of cancer. Alternatively stated, the onset of cancer in thesubject may be reduced in likelihood or probability and/or delayed.

In particular embodiments, cells may be removed from a subject withcancer and contacted with a virus vector expressing a cancer cellantigen according to the instant disclosure. The modified cell is thenadministered to the subject, whereby an immune response against thecancer cell antigen is elicited. This method can be advantageouslyemployed with immunocompromised subjects that cannot mount a sufficientimmune response in vivo (i.e., cannot produce enhancing antibodies insufficient quantities).

It is known in the art that immune responses may be enhanced byimmunomodulatory cytokines (e.g., alpha-interferon, beta-interferon,gamma-interferon, omega-interferon, tau-interferon, interleukin-1-alpha,interleukin-113, interleukin-2, interleukin-3, interleukin-4,interleukin 5, interleukin-6, interleukin-7, interleukin-8,interleukin-9, interleukin-10, interleukin-11, interleukin-12,interleukin-13, interleukin-14, interleukin-18, B cell Growth factor,CD40 Ligand, tumor necrosis factor-alpha, tumor necrosis factor-β,monocyte chemoattractant protein-1, granulocyte-macrophage colonystimulating factor, and lymphotoxin). Accordingly, immunomodulatorycytokines (preferably, CTL inductive cytokines) may be administered to asubject in conjunction with the virus vector. Cytokines may beadministered by any method known in the art. Exogenous cytokines may beadministered to the subject, or alternatively, a nucleic acid encoding acytokine may be delivered to the subject using a suitable vector, andthe cytokine produced in vivo.

Subjects, Pharmaceutical Formulations, and Modes of Administration

Virus vectors and capsids according to the present disclosure find usein both veterinary and medical applications. Suitable subjects includeboth avians and mammals. The term “avian” as used herein includes, butis not limited to, chickens, ducks, geese, quail, turkeys, pheasant,parrots, parakeets, and the like. The term “mammals” as used hereinincludes, but is not limited to, humans, non-human primates, bovines,ovines, caprines, equines, felines, canines, lagomorphs, etc. Humansubjects include neonates, infants, juveniles, adults and geriatricsubjects. In some embodiments, a human subject can be less than 6 monthsold, less than 2 years old, less than 5 years old, less than 10 yearsold, 10-18 years old, 19-29 years old, 30-35 years old, 36-40 years old,or older than 40 years old.

In representative embodiments, the subject is “in need” of the methodsdescribed herein.

In particular embodiments, a pharmaceutical composition is providedcomprising a virus vector and/or capsid and/or capsid protein and/orvirus particle of the disclosure in a pharmaceutically acceptablecarrier and, optionally, other medicinal agents, pharmaceutical agents,stabilizing agents, buffers, carriers, adjuvants, diluents, etc. Forinjection, the carrier will typically be a liquid. For other methods ofadministration, the carrier may be either solid or liquid. Forinhalation administration, the carrier will be respirable, andoptionally can be in solid or liquid particulate form.

By “pharmaceutically acceptable” it is meant a material that is nottoxic or otherwise undesirable, i.e., the material may be administeredto a subject without causing any undesirable biological effects.

One aspect of the present disclosure is a method of transferring anucleic acid to a cell in vitro. The virus vector may be introduced intothe cells at the appropriate multiplicity of infection according tostandard transduction methods suitable for the particular target cells.Titers of virus vector to administer can vary, depending upon the targetcell type and number, and the particular virus vector, and can bedetermined by those of skill in the art without undue experimentation.In representative embodiments, at least about 10³ infectious units,optionally at least about 10⁵ infectious units are introduced to thecell.

The cell(s) into which the virus vector is introduced can be of anytype, including but not limited to neural cells (including cells of theperipheral and central nervous systems, in particular, brain cells suchas neurons and oligodendrocytes), lung cells, cells of the eye(including retinal cells, retinal pigment epithelium, and cornealcells), epithelial cells (e.g., gut and respiratory epithelial cells),muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, smoothmuscle cells and/or diaphragm muscle cells), dendritic cells, pancreaticcells (including islet cells), hepatic cells, myocardial cells, bonecells (e.g., bone marrow stem cells), hematopoietic stem cells, spleencells, keratinocytes, fibroblasts, endothelial cells, prostate cells,germ cells, and the like. In representative embodiments, the cell can beany progenitor cell. As a further possibility, the cell can be a stemcell (e.g., neural stem cell, liver stem cell). As still a furtheralternative, the cell can be a cancer or tumor cell. Moreover, the cellcan be from any species of origin, as indicated above.

The virus vector can be introduced into cells in vitro for the purposeof administering the modified cell to a subject. In particularembodiments, the cells have been removed from a subject, the virusvector is introduced therein, and the cells are then administered backinto the subject. Methods of removing cells from subject formanipulation ex vivo, followed by introduction back into the subject areknown in the art (see, e.g., U.S. Pat. No. 5,399,346). Alternatively,the recombinant virus vector can be introduced into cells from a donorsubject, into cultured cells, or into cells from any other suitablesource, and the cells are administered to a subject in need thereof(i.e., a “recipient” subject).

Suitable cells for ex vivo nucleic acid delivery are as described above.Dosages of the cells to administer to a subject will vary upon the age,condition and species of the subject, the type of cell, the nucleic acidbeing expressed by the cell, the mode of administration, and the like.Typically, at least about 10² to about 10⁸ cells or at least about 10³to about 10⁶ cells will be administered per dose in a pharmaceuticallyacceptable carrier. In particular embodiments, the cells transduced withthe virus vector are administered to the subject in a therapeuticallyeffective amount in combination with a pharmaceutical carrier.

In some embodiments, the virus vector is introduced into a cell and thecell can be administered to a subject to elicit an immunogenic responseagainst the delivered polypeptide (e.g., expressed as a transgene or inthe capsid). Typically, a quantity of cells expressing animmunogenically effective amount of the polypeptide in combination witha pharmaceutically acceptable carrier is administered. An“immunogenically effective amount” is an amount of the expressedpolypeptide that is sufficient to evoke an active immune responseagainst the polypeptide in the subject to which the pharmaceuticalformulation is administered. In particular embodiments, the dosage issufficient to produce a protective immune response (as defined above).The degree of protection conferred need not be complete or permanent, aslong as the benefits of administering the immunogenic polypeptideoutweigh any disadvantages thereof.

Thus, the present disclosure provides a method of administering anucleic acid to a cell, the method comprising contacting the cell withthe virus vector, virus particle and/or composition of this disclosure.

A further aspect of the disclosure is a method of administering thevirus vector, virus particle and/or virus capsid of this disclosure to asubject. Thus, the present disclosure also provides a method ofdelivering a nucleic acid to a subject, comprising administering to thesubject a virus particle, virus vector and/or composition of thisdisclosure. Administration of the virus vectors, virus particles and/orcapsids according to the present disclosure to a human subject or ananimal in need thereof can be by any means known in the art. Optionally,the virus vector, virus particle and/or capsid is delivered in atherapeutically effective dose in a pharmaceutically acceptable carrier.In preferred embodiments, a therapeutically effective amount of thevirus vector, virus particle and/or capsid is delivered.

The virus vectors and/or capsids of the disclosure can further beadministered to elicit an immunogenic response (e.g., as a vaccine).Typically, immunogenic compositions of the present disclosure comprisean immunogenically effective amount of virus vector and/or capsid incombination with a pharmaceutically acceptable carrier. Optionally, thedosage is sufficient to produce a protective immune response (as definedabove). The degree of protection conferred need not be complete orpermanent, as long as the benefits of administering the immunogenicpolypeptide outweigh any disadvantages thereof. Subjects and immunogensare as described above.

Dosages of the virus vector and/or capsid to be administered to asubject depend upon the mode of administration, the disease or conditionto be treated and/or prevented, the individual subject's condition, theparticular virus vector or capsid, and the nucleic acid to be delivered,and the like, and can be determined in a routine manner. Exemplary dosesfor achieving therapeutic effects are titers of at least about 10⁵,about 10⁶, about 10⁷, about 10⁸, about 10⁹, about 10¹⁰, about 10¹¹,about 10¹², about 10¹³, about 10¹⁴, about 10¹⁵ transducing units,optionally about 10⁸-10¹³ transducing units.

In particular embodiments, more than one administration (e.g., two,three, four or more administrations) may be employed to achieve thedesired level of gene expression over a period of various intervals,e.g., daily, weekly, monthly, yearly, etc.

Exemplary modes of administration include oral, rectal, transmucosal,intranasal, inhalation (e.g., via an aerosol), buccal (e.g.,sublingual), vaginal, intrathecal, intraocular, transdermal, in utero(or in ovo), parenteral (e.g., intravenous, subcutaneous, intradermal,intramuscular [including administration to skeletal, diaphragm and/orcardiac muscle], intradermal, intrapleural, intracerebral, andintraarticular), topical (e.g., to both skin and mucosal surfaces,including airway surfaces, and transdermal administration),intralymphatic, and the like, as well as direct tissue or organinjection (e.g., to liver, skeletal muscle, cardiac muscle, diaphragmmuscle or brain). Administration can also be to a tumor (e.g., in ornear a tumor or a lymph node). In some embodiments, the virus vector orcomposition is administered to the eye of the subject, for example viaan intravitreal, subretinal, subconjuctival, retrobulbar, intracameraland/or suprachoroidal route. The most suitable route in any given casewill depend on the nature and severity of the condition being treatedand/or prevented and on the nature of the particular vector that isbeing used.

Administration to skeletal muscle according to the present disclosureincludes but is not limited to administration to skeletal muscle in thelimbs (e.g., upper arm, lower arm, upper leg, and/or lower leg), back,neck, head (e.g., tongue), thorax, abdomen,

pelvis/perineum, and/or digits. Suitable skeletal muscles include butare not limited to abductor digiti minimi (in the hand), abductor digitiminimi (in the foot), abductor hallucis, abductor ossis metatarsiquinti, abductor pollicis brevis, abductor pollicis longus, adductorbrevis, adductor hallucis, adductor longus, adductor magnus, adductorpollicis, anconeus, anterior scalene, articularis genus, biceps brachii,biceps femoris, brachialis, brachioradialis, buccinator,coracobrachialis, corrugator supercilii, deltoid, depressor anguli oris,depressor labii inferioris, digastric, dorsal interossei (in the hand),dorsal interossei (in the foot), extensor carpi radialis brevis,extensor carpi radialis longus, extensor carpi ulnaris, extensor digitiminimi, extensor digitorum, extensor digitorum brevis, extensordigitorum longus, extensor hallucis brevis, extensor hallucis longus,extensor indicis, extensor pollicis brevis, extensor pollicis longus,flexor carpi radialis, flexor carpi ulnaris, flexor digiti minimi brevis(in the hand), flexor digiti minimi brevis (in the foot), flexordigitorum brevis, flexor digitorum longus, flexor digitorum profundus,flexor digitorum superficialis, flexor hallucis brevis, flexor hallucislongus, flexor pollicis brevis, flexor pollicis longus, frontalis,gastrocnemius, geniohyoid, gluteus maximus, gluteus medius, gluteusminimus, gracilis, iliocostalis cervicis, iliocostalis lumborum,iliocostalis thoracis, illiacus, inferior gemellus, inferior oblique,inferior rectus, infraspinatus, interspinalis, intertransversi, lateralpterygoid, lateral rectus, latissimus dorsi, levator anguli oris,levator labii superioris, levator labii superioris alaeque nasi, levatorpalpebrae superioris, levator scapulae, long rotators, longissimuscapitis, longissimus cervicis, longissimus thoracis, longus capitis,longus colli, lumbricals (in the hand), lumbricals (in the foot),masseter, medial pterygoid, medial rectus, middle scalene, multifidus,mylohyoid, obliquus capitis inferior, obliquus capitis superior,obturator externus, obturator internus, occipitalis, omohyoid, opponensdigiti minimi, opponens pollicis, orbicularis oculi, orbicularis oris,palmar interossei, palmaris brevis, palmaris longus, pectineus,pectoralis major, pectoralis minor, peroneus brevis, peroneus longus,peroneus tertius, piriformis, plantar interossei, plantaris, platysma,popliteus, posterior scalene, pronator quadratus, pronator teres, psoasmajor, quadratus femoris, quadratus plantae, rectus capitis anterior,rectus capitis lateralis, rectus capitis posterior major, rectus capitisposterior minor, rectus femoris, rhomboid major, rhomboid minor,risorius, sartorius, scalenus minimus, semimembranosus, semispinaliscapitis, semispinalis cervicis, semispinalis thoracis, semitendinosus,serratus anterior, short rotators, soleus, spinalis capitis, spinaliscervicis, spinalis thoracis, splenius capitis, splenius cervicis,sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius,subscapularis, superior gemellus, superior oblique, superior rectus,supinator, supraspinatus, temporalis, tensor fascia lata, teres major,teres minor, thoracis, thyrohyoid, tibialis anterior, tibialisposterior, trapezius, triceps brachii, vastus intermedius, vastuslateralis, vastus medialis, zygomaticus major, and zygomaticus minor,and any other suitable skeletal muscle as known in the art.

The virus vector and/or capsid can be delivered to skeletal muscle byintravenous administration, intra-arterial administration,intraperitoneal administration, limb perfusion, (optionally, isolatedlimb perfusion of a leg and/or arm; see, e.g. Arruda et al., (2005)Blood 105: 3458-3464), and/or direct intramuscular injection. Inparticular embodiments, the virus vector and/or capsid is administeredto a limb (arm and/or leg) of a subject (e.g., a subject with musculardystrophy such as DMD) by limb perfusion, optionally isolated limbperfusion (e.g., by intravenous or intra-articular administration). Inembodiments of the disclosure, the virus vectors and/or capsids of thedisclosure can advantageously be administered without employing“hydrodynamic” techniques. Tissue delivery (e.g., to muscle) of priorart vectors is often enhanced by hydrodynamic techniques (e.g.,intravenous/intravenous administration in a large volume), whichincrease pressure in the vasculature and facilitate the ability of thevector to cross the endothelial cell barrier. In particular embodiments,the viral vectors and/or capsids of the disclosure can be administeredin the absence of hydrodynamic techniques such as high volume infusionsand/or elevated intravascular pressure (e.g., greater than normalsystolic pressure, for example, less than or equal to a 5%, 10%, 15%,20%, 25% increase in intravascular pressure over normal systolicpressure). Such methods may reduce or avoid the side effects associatedwith hydrodynamic techniques such as edema, nerve damage and/orcompartment syndrome. Administration to cardiac muscle includesadministration to the left atrium, right atrium, left ventricle, rightventricle and/or septum. The virus vector and/or capsid can be deliveredto cardiac muscle by intravenous administration, intra-arterialadministration such as intra-aortic administration, direct cardiacinjection (e.g., into left atrium, right atrium, left ventricle, rightventricle), and/or coronary artery perfusion.

Administration to diaphragm muscle can be by any suitable methodincluding intravenous administration, intra-arterial administration,and/or intra-peritoneal administration.

Delivery to a target tissue can also be achieved by delivering a depotcomprising the virus vector and/or capsid. In representativeembodiments, a depot comprising the virus vector and/or capsid isimplanted into skeletal, cardiac and/or diaphragm muscle tissue or thetissue can be contacted with a film or other matrix comprising the virusvector and/or capsid. Such implantable matrices or substrates aredescribed in U.S. Pat. No. 7,201,898.

In particular embodiments, a virus vector and/or virus capsid accordingto the present disclosure is administered to skeletal muscle, diaphragmmuscle and/or cardiac muscle (e.g., to treat and/or prevent musculardystrophy, heart disease [for example, PAD or congestive heartfailure]).

In representative embodiments, the disclosure is used to treat and/orprevent disorders of skeletal, cardiac and/or diaphragm muscle.

In a representative embodiment, a method of treating and/or preventingmuscular dystrophy in a subject in need thereof is provided, the methodcomprising: administering a treatment or prevention effective amount ofa virus vector of the disclosure to a mammalian subject, wherein thevirus vector comprises a heterologous nucleic acid encoding dystrophin,a mini-dystrophin, a micro-dystrophin, myostatin propeptide,follistatin, activin type II soluble receptor, IGF-1, anti-inflammatorypolypeptides such as the Ikappa B dominant mutant, sarcospan, utrophin,a micro-dystrophin, laminin-a2, alpha-sarcoglycan, beta-sarcoglycan,gamma-sarcoglycan, delta-sarcoglycan, IGF-1, an antibody or antibodyfragment against myostatin or myostatin propeptide, and/or RNAi againstmyostatin. In particular embodiments, the virus vector can beadministered to skeletal, diaphragm and/or cardiac muscle as describedelsewhere herein.

Alternatively, the disclosure can be practiced to deliver a nucleic acidto skeletal, cardiac or diaphragm muscle, which is used as a platformfor production of a polypeptide (e.g., an enzyme) or functional RNA(e.g., RNAi, micro RNA, antisense RNA) that normally circulates in theblood or for systemic delivery to other tissues to treat and/or preventa disorder (e.g., a metabolic disorder, such as diabetes [e.g.,insulin], hemophilia [e.g., Factor IX or Factor VIII], amucopolysaccharide disorder [e.g., Sly syndrome, Hurler Syndrome, ScheieSyndrome, Hurler-Scheie Syndrome, Hunter's Syndrome, Sanfilippo SyndromeA, B, C, D, Morquio Syndrome, Maroteaux-Lamy Syndrome, etc.] or alysosomal storage disorder such as Gaucher's disease[glucocerebrosidase] or Fabry disease [a-galactosidase A] or a glycogenstorage disorder such as Pompe disease [lysosomal acid alphaglucosidase]). Other suitable proteins for treating and/or preventingmetabolic disorders are described herein. The use of muscle as aplatform to express a nucleic acid of interest is described in U.S.Patent Pub. No. US 2002/0192189.

Thus, as one aspect, the disclosure further encompasses a method oftreating and/or preventing a metabolic disorder in a subject in needthereof, the method comprising: administering a treatment or preventioneffective amount of a virus vector of the disclosure to skeletal muscleof a subject, wherein the virus vector comprises a heterologous nucleicacid encoding a polypeptide, wherein the metabolic disorder is a resultof a deficiency and/or defect in the polypeptide. Illustrative metabolicdisorders and heterologous nucleic acids encoding polypeptides aredescribed herein. Optionally, the polypeptide is secreted (e.g., apolypeptide that is a secreted polypeptide in its native state or thathas been engineered to be secreted, for example, by operable associationwith a secretory signal sequence as is known in the art). Without beinglimited by any particular theory of the disclosure, according to thisembodiment, administration to the skeletal muscle can result insecretion of the polypeptide into the systemic circulation and deliveryto target tissue(s). Methods of delivering virus vectors to skeletalmuscle is described in more detail herein.

The disclosure can also be practiced to produce noncoding RNA, such asantisense RNA, RNAi or other functional RNA (e.g., a ribozyme) forsystemic delivery.

The disclosure also provides a method of treating and/or preventingcongenital heart failure or PAD in a subject in need thereof, the methodcomprising administering a treatment or prevention effective amount of avirus vector of the disclosure to a mammalian subject, wherein the virusvector comprises a heterologous nucleic acid encoding, for example, asarcoplasmic endoreticulum Ca²⁺-ATPase (SERCA2a), an angiogenic factor,phosphatase inhibitor 1 (1-1) and fragments thereof (e.g., 11C), RNAiagainst phospholamban; a phospholamban inhibitory or dominant-negativemolecule such as phospholamban S16E, a zinc finger protein thatregulates the phospholamban gene, beta-2-adrenergic receptor,beta-2-adrenergic receptor kinase (BARK), PI3 kinase, calsarcan, aβ-adrenergic receptor kinase inhibitor (PARKct), inhibitor 1 of proteinphosphatase 1 and fragments thereof (e.g., I1 C), S100A1, parvalbumin,adenylyl cyclase type 6, a molecule that effects G-protein coupledreceptor kinase type 2 knockdown such as a truncated constitutivelyactive bARKct, Pim-1, PGC-I α, SOD-1, SOD-2, EC-SOD, kallikrein. HIF,thymosin-p4, mir-1, mir-133, mir-206, mir-208 and/or mir-26a.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Alternatively,one may administer the virus vector and/or virus capsids of thedisclosure in a local rather than systemic manner, for example, in adepot or sustained-release formulation. Further, the virus vector and/orvirus capsid can be delivered adhered to a surgically implantable matrix(e.g., as described in U.S. Patent Publication No. US-2004-0013645-A1).

The virus vectors and/or virus capsids disclosed herein can beadministered to the lungs of a subject by any suitable means, optionallyby administering an aerosol suspension of respirable particles comprisedof the virus vectors and/or virus capsids, which the subject inhales.The respirable particles can be liquid or solid. Aerosols of liquidparticles comprising the virus vectors and/or virus capsids may beproduced by any suitable means, such as with a pressure-driven aerosolnebulizer or an ultrasonic nebulizer, as is known to those of skill inthe art. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particlescomprising the virus vectors and/or capsids may likewise be producedwith any solid particulate medicament aerosol generator, by techniquesknown in the pharmaceutical art.

The virus vectors and virus capsids can be administered to tissues ofthe CNS (e.g., brain, eye) and may advantageously result in broaderdistribution of the virus vector or capsid than would be observed in theabsence of the present disclosure.

In particular embodiments, the delivery vectors described herein may beadministered to treat diseases of the CNS, including genetic disorders,neurodegenerative disorders, psychiatric disorders and tumors.Illustrative diseases of the CNS include, but are not limited toAdrenomyeloneuropathy (AMN), Alzheimer's disease, Angelman Syndrome,Parkinson's disease, Huntington's disease, Canavan disease, Leigh'sdisease, Refsum disease, Tourette syndrome, primary lateral sclerosis,amyotrophic lateral sclerosis, progressive muscular atrophy, Pick'sdisease, muscular dystrophy, multiple sclerosis, myasthenia gravis,Binswanger's disease, trauma due to spinal cord or head injury, TaySachs disease, GM2 Gangliosidosis, Lesch-Nyhan disease, MC4R Obesity,Metachromatic Leukodystrophy (MLD), MPS I (Hurler/Scheie), MPS IIIA(Sanfilippo A), Niemann Pick C1, Rett Syndrome, Spinal Muscular Atrophy(SMA), AADC Deficiency, Monogenic Amyotropic Lateral Sclerosis (ALS),Alpha mannosidosis, Alzheimer's Disease, Aspartylglucosaminuria, DravetSyndrome, Giant Axonal Neuropathy, Globoid Cell Leukodystrophy (Krabbe),Glut 1 Deficiency, GM1 Gangliosidosis, Infantile Neuronal CeroidLipfuscinosis (INCL, Batten), Juvenile Neuronal Ceroid Lipfuscinosis(JNCL, Batten), Late Infantile Neuronal Ceroid Lipfuscinosis (LINCL,Batten), MPS II (Hunter), MPS IIIB (Sanfilippo B), MPS IIIC (SanfilippoC), MPS IVA (Morquio Syndrome), MPS VI (Maroteaux-Lamy), PeroxisomeBiogenesis Disorders (Zellweger Syndrome Spectrum), Sandhoff Disease(GM2 Gangliosidosis), Lesch-Nyhan disease, epilepsy, cerebral infarcts,psychiatric disorders including mood disorders (e.g., depression,bipolar affective disorder, persistent affective disorder, secondarymood disorder), schizophrenia, drug dependency (e.g., alcoholism andother substance dependencies), neuroses (e.g., anxiety, obsessionaldisorder, somatoform disorder, dissociative disorder, grief, post-partumdepression), psychosis (e.g., hallucinations and delusions), dementia,paranoia, attention deficit disorder, psychosexual disorders, sleepingdisorders, pain disorders, eating or weight disorders (e.g., obesity,cachexia, anorexia nervosa, and bulemia) and cancers and tumors (e.g.,pituitary tumors) of the CNS.

Disorders of the CNS may include ophthalmic disorders involving theretina, posterior tract, and optic nerve. For example, such disordersmay include retinitis pigmentosa, diabetic retinopathy and other retinaldegenerative diseases, uveitis, age-related macular degeneration, opticneuritis, Leber's congenital amaurosis, Leber's hereditary opticneuropathy, achromatopsia, X-linked retinoschisis, optic neuritis,choroideremia, optic atrophy, retinal cone dystrophy, retinopathy,retinoblastoma, glaucoma, Bardet-Biedl syndrome, Usher syndrome,aniridia, Friedreich's ataxia, vitelliform macular dystrophy,retinoblastoma, Stargardt disease, Charcot-Marie-Tooth disease, Fuch'sdystrophy, propionic acidemia, or color blindness.

Thus, in some embodiments, the present disclosure provides a method oftreating an ophthalmic disorder or defect in a subject, comprisingadministering to the subject a viral vector of this disclosure, whereinthe viral vector comprises a nucleic acid molecule that encodes atherapeutic protein or therapeutic RNA effective in treating theophthalmic disorder or defect. In some embodiments of this method, theviral vector is administered via an intravitreal, subretinal,subconjuctival, retrobulbar, intracameral, and/or suprachoroidal route.

Most, if not all, ophthalmic diseases and disorders are associated withone or more of three types of indications: (1) angiogenesis, (2)inflammation, and (3) degeneration. The delivery vectors of the presentdisclosure can be employed to deliver anti-angiogenic factors;anti-inflammatory factors; factors that retard cell degeneration,promote cell sparing, or promote cell growth and combinations of theforegoing.

Diabetic retinopathy, for example, is characterized by angiogenesis.Diabetic retinopathy can be treated by delivering one or moreanti-angiogenic factors either intraocularly (e.g., in the vitreous) orperiocularly (e.g., in the sub-Tenon's region). One or more neurotrophicfactors may also be co-delivered, either intraocularly (e.g.,intravitreally) or periocularly.

Uveitis involves inflammation. One or more anti-inflammatory factors canbe administered by intraocular (e.g., vitreous or anterior chamber)administration of a delivery vector of the disclosure.

Retinitis pigmentosa, by comparison, is characterized by retinaldegeneration. In representative embodiments, retinitis pigmentosa can betreated by intraocular (e.g., vitreal administration) of a deliveryvector encoding one or more neurotrophic factors.

Age-related macular degeneration involves both angiogenesis and retinaldegeneration. This disorder can be treated by administering the deliveryvectors encoding one or more neurotrophic factors intraocularly (e.g.,vitreous) and/or one or more anti-angiogenic factors intraocularly orperiocularly (e.g., in the sub-Tenon's region).

Glaucoma is characterized by increased ocular pressure and loss ofretinal ganglion cells. Treatments for glaucoma include administrationof one or more neuroprotective agents that protect cells fromexcitotoxic damage using the delivery vectors. Such agents includeN-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophicfactors, delivered intraocularly, optionally intravitreally.

In other embodiments, the present disclosure may be used to treatseizures, e.g., to reduce the onset, incidence or severity of seizures.The efficacy of a therapeutic treatment for seizures can be assessed bybehavioral (e.g., shaking, ticks of the eye or mouth) and/orelectrographic means (most seizures have signature electrographicabnormalities). Thus, the disclosure can also be used to treat epilepsy,which is marked by multiple seizures over time.

In one representative embodiment, somatostatin (or an active fragmentthereof) is administered to the brain using a delivery vector of thedisclosure to treat a pituitary tumor. According to this embodiment, thedelivery vector encoding somatostatin (or an active fragment thereof) isadministered by microinfusion into the pituitary. Likewise, suchtreatment can be used to treat acromegaly (abnormal growth hormonesecretion from the pituitary). The nucleic acid (e.g., GenBank AccessionNo. J00306) and amino acid (e.g., GenBank Accession No. P01166; containsprocessed active peptides somatostatin-28 and somatostatin-14) sequencesof somatostatins are known in the art.

In particular embodiments, the vector can comprise a secretory signal asdescribed in U.S. Pat. No. 7,071,172.

In representative embodiments of the disclosure, the virus vector and/orvirus capsid is administered to the CNS (e.g., to the brain or to theeye). The virus vector and/or capsid may be introduced into the spinalcord, brainstem (medulla oblongata, pons), midbrain (hypothalamus,thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland),cerebellum, telencephalon (corpus striatum, cerebrum including theoccipital, temporal, parietal and frontal lobes, cortex, basal ganglia,hippocampus and portaamygdala), limbic system, neocortex, corpusstriatum, cerebrum, and inferior colliculus. The virus vector and/orcapsid may also be administered to different regions of the eye such asthe retina, cornea and/or optic nerve.

The virus vector and/or capsid may be delivered into the cerebrospinalfluid (e.g., by lumbar puncture) for more disperse administration of thedelivery vector. The virus vector and/or capsid may further beadministered intravascularly to the CNS in situations in which theblood-brain barrier has been perturbed (e.g., brain tumor or cerebralinfarct).

The virus vector and/or capsid can be administered to the desiredregion(s) of the CNS by any route known in the art, including but notlimited to, intrathecal, intra-ocular, intracerebral, intraventricular,intravenous (e.g., in the presence of a sugar such as mannitol),intranasal, intra-aural, intra-ocular (e.g., intra-vitreous,sub-retinal, anterior chamber) and peri-ocular (e.g., sub-Tenon'sregion) delivery as well as intramuscular delivery with retrogradedelivery to motor neurons. In particular embodiments, the virus vectorand/or capsid is administered in a liquid formulation by directinjection (e.g., stereotactic injection) to the desired region orcompartment in the CNS. In other embodiments, the virus vector and/orcapsid may be provided by topical application to the desired region orby intra-nasal administration of an aerosol formulation. Administrationto the eye, may be by topical application of liquid droplets. As afurther alternative, the virus vector and/or capsid may be administeredas a solid, slow-release formulation (see, e.g., U.S. Pat. No.7,201,898).

In yet additional embodiments, the virus vector can used for retrogradetransport to treat and/or prevent diseases and disorders involving motorneurons (e.g., amyotrophic lateral sclerosis (ALS); spinal muscularatrophy (SMA), etc.). For example, the virus vector can be delivered tomuscle tissue from which it can migrate into neurons.

The following examples, which are included herein for illustrationpurposes only, are not intended to be limiting.

NUMBERED EMBODIMENTS

The following numbered embodiments are included within the scope of thedisclosure.

1. A recombinant adeno-associated virus (AAV) capsid protein, whereinthe capsid protein comprises a substitution in a surface-exposed loop ofthe AAV capsid protein, wherein the substitution has a sequence of anyone of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 187, 188, 189,190, 191, 192, 193, or 194.

2. The recombinant AAV capsid protein of embodiment 1, wherein thesubstitution has a sequence of any one of SEQ ID NO: 14, 17, 19, or 22.

3. The recombinant AAV capsid protein of embodiment 1 or 2, wherein thecapsid protein comprises an amino acid sequence with at least 80%sequence identity to a capsid protein of any one of AAV1, AAV2, AAV3,AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh.8,AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV or avian AAV.

4. The recombinant AAV capsid protein of any one of embodiments 1-3,wherein the capsid protein comprises an amino acid sequence that has atleast 90% sequence identity with any one of SEQ ID NO: 11-12, 23-49, or195-254.

5. The recombinant AAV capsid protein of any one of embodiments 1-3,wherein the capsid comprises an amino acid sequence of any one of SEQ IDNO: 11-12, 23-49, or 195-254.

6. The recombinant AAV capsid protein of embodiment 5, wherein thecapsid comprises an amino acid sequence of any one of SEQ ID NO. 32, 35,37, or 40.

7. The recombinant AAV capsid protein of any one of embodiments 1-3,wherein the amino acid substitution replaces the amino acids in theregion corresponding to amino acid positions 591-596 of SEQ ID NO. 11 oramino acids 590-595 of SEQ ID NO. 12.

8. A recombinant AAV capsid protein, wherein the capsid proteincomprises a substitution comprising a sequence of six amino acids(X¹-X²-X³-X⁴-X⁵-X⁶) that does not occur in the native AAV capsidprotein, wherein X² is V and X⁵ is L (SEQ ID NO: 186).

9. The recombinant AAV capsid protein of embodiment 8, wherein thesubstitution replaces the amino acids in the region corresponding toamino acid positions 591-596 of SEQ ID NO. 11 or amino acids 590-595 ofSEQ ID NO. 12.

10. The recombinant AAV capsid protein of embodiment 8 or 9, wherein thesubstitution is in a surface-exposed loop of the AAV capsid protein.

11. The recombinant AAV capsid protein of any one of embodiments 8-10,wherein X¹ is not T, X³ is not D, X⁴ is not R, and X⁶ is not T.

12. The recombinant AAV capsid protein of any one of embodiments 8-11,wherein X¹ is K, G, F, I, H, or R.

13. The recombinant AAV capsid protein of any one of embodiments 8-12,wherein X³ is R, L, H, G, or N.

14. The recombinant AAV capsid protein of any one of embodiments 8-13,wherein X⁴ is D, A, S, or V, or R.

15. The recombinant AAV capsid protein of any one of embodiments 8-14,wherein X⁶ is F, R, P, N, or Q.

16. The recombinant AAV capsid protein of any one of embodiments 8-11,wherein X¹ is K, X³ is R, X⁴ is D, and X⁶ is F.

17. The recombinant AAV capsid protein of any one of embodiments 8-11,wherein X¹ is R, X³ is L, X⁴ is A, and X⁶ is R.

18. The recombinant AAV capsid protein of any one of embodiments 8-11,wherein X¹ is R, X³ is H, X⁴ is A, and X⁶ is R.

19. The recombinant AAV capsid protein of any one of embodiments 8-11,wherein X¹ is R, X³ is H, X⁴ is S, and X⁶ is R.

20. The recombinant AAV capsid protein of any one of embodiments 8-11,wherein X¹ is G, X³ is G, X⁴ is V, and X⁶ is P.

21. The recombinant AAV capsid protein of any one of embodiments 8-11,wherein X¹ is F, X³ is N, X⁴ is A, and X⁶ is N.

22. The recombinant AAV capsid protein of any one of embodiments 8-11,wherein X¹ is I, X³ is R, X⁴ is S, and X⁶ is N.

23. The recombinant AAV capsid protein of any one of embodiments 8-11,wherein X¹ is H, X³ is L, X⁴ is R, and X⁶ is N.

24. The recombinant AAV capsid protein of any one of embodiments 8-11,wherein X¹ is R, X³ is L, X⁴ is A, and X⁶ is Q.

25. The recombinant AAV capsid protein of any one of embodiments 8-24wherein the AAV capsid protein is of an AAV serotype selected from AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12,AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV and avian AAV.

26. A recombinant AAV capsid protein comprising the amino acid sequenceof any one of SEQ ID NO: 11-12, 23-49, or 195-254.

27. The recombinant AAV capsid protein of embodiment 26 comprising theamino acid sequence of SEQ ID NO: 32, 35, 37, or 40.

28. A nucleotide sequence encoding an AAV capsid protein of any one ofembodiments 1-27.

29. The nucleotide sequence of embodiment 28, wherein the nucleotidesequence is a DNA sequence.

30. The nucleotide sequence of embodiment 28, wherein the nucleotidesequence is an RNA sequence.

31. An expression vector comprising the nucleotide sequence of any oneof embodiments 28-30.

32. A cell comprising the nucleotide sequence of any one of embodiments28 to 30 or the expression vector of embodiment 31.

33. A recombinant AAV viral vector comprising the capsid protein of anyone of embodiments 1 to 27.

34. The recombinant AAV viral vector of embodiment 33, wherein the AAVviral vector has an enhanced transduction profile with respect to atarget tissue, as compared to an AAV viral vector comprising a capsidprotein that does not comprise the substitution.

35. The recombinant AAV viral vector of embodiment 34, wherein thetarget tissue is an ophthalmic tissue.

36. The recombinant AAV viral vector of any one of embodiments 33-35,further comprising a cargo nucleic acid encapsidated by the capsidprotein.

37. The recombinant AAV viral vector of embodiment 36, wherein the cargonucleic acid encodes a therapeutic protein or RNA.

38. The recombinant AAV viral vector of embodiment 36 or 37, wherein thecargo nucleic acid encodes one or more of the following proteins or anantibody that binds thereto: cystic fibrosis transmembrane regulatorprotein (CFTR), dystrophin, myostatin propeptide, follistatin, activintype 11 soluble receptor, IGF-I, Ikappa B dominant mutant, sarcospan,utrophin, mini-utrophin, Factor VIII, Factor IX, Factor X,erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase,superoxide dismutase, leptin, the LDL receptor, lipoprotein lipase,ornithine transcarbamylase, β-globin, α-globin. spectrin,alpha-1-antitrypsin, adenosine deaminase, hypoxanthine guaninephosphoribosyl transferase, β-glucocerebrosidase, sphingomyelinase,lysosomal hexosaminidase A, branched-chain keto acid dehydrogenase,RPE65 protein, alpha-interferon, beta-interferon, gamma-interferon,interleukin-2, interleukin-4, granulocyte-macrophage colony stimulatingfactor, lymphotoxin, a peptide growth factors, a neurotrophic factors,somatotropin, insulin, insulin-like growth factors 1 or 2, plateletderived growth factor, epidermal growth factor, fibroblast growthfactor, nerve growth factor, neurotrophic factor -3 or -4, brain-derivedneurotrophic factor, RANKL, VEGF, glial derived growth factor,transforming growth factor-alpha or beta, lysosomal acidalpha-glucosidase, alpha-galactosidase A, tumor necrosis growth factorsoluble receptor, S100A1, parvalbumin, adenylyl cyclase type 6, SERCA2A,Inhibitor 1 of PP 1 or fragments thereof, truncated constitutivelyactive bARKct, IRAP, anti-myostatin protein, aspartoacylase,trastuzumab, galanin, Neuropeptide Y, Vasohibin 2, thymidine kinase,cytosine deaminase, diphtheria toxin, tumor necrosis factor, p53, Rb,Wt-1, TRAIL, RS1, opsin, TKR-beta, C3, CFH, and/or FAS-ligand.

39. The recombinant AAV viral vector of embodiment 36 or 37, wherein thecargo nucleic acid encodes a gene-editing molecule.

40. The recombinant AAV viral vector of embodiment 39, wherein thegene-editing molecule is a nuclease.

41. The recombinant AAV viral vector of embodiment 39 or 40, wherein thegene-editing molecule is a Cas9 nuclease.

42. The recombinant AAV viral vector of embodiment 39 or 40, wherein thegene-editing molecule is a Cpf1 nuclease.

43. The recombinant AAV viral vector of embodiment 39, wherein thegene-editing molecule is a guide RNA.

44. A pharmaceutical composition comprising the recombinant AAV viralvector of any one of embodiments 33 to 43.

45. The pharmaceutical composition of claim 44, wherein the compositionfurther comprises a pharmaceutically acceptable carrier.

46. A method of treating a patient in need thereof comprisingadministering to the patient a therapeutically effective amount of arecombinant AAV viral vector of any one of embodiments 33 to 43 or thepharmaceutical composition of embodiment 44 or 45.

47. The method of embodiment 46, wherein the patient has a disease ordisorder of the eye.

48. The method of embodiment 47, wherein the disease or disorder of theeye is retinitis pigmentosa, macular degeneration, optic neuritis,Leber's congenital amaurosis, Leber's hereditary optic neuropathy,achromatopsia, X-linked retinoschisis, optic neuritis, choroideremia,optic atrophy, retinal cone dystrophy, retinopathy, retinoblastoma,glaucoma, Bardet-Biedl syndrome, Usher syndrome, aniridia, Friedreich'sataxia, vitelliform macular dystrophy, retinoblastoma, Stargardtdisease, Charcot-Marie-Tooth disease, Fuch's dystrophy, propionicacidemia, or color blindness.

49. The method of any one of embodiments 46-48, wherein the patient is amammal.

50. The method of embodiment 49, wherein the patient is a human.

51. A method of introducing a nucleic acid molecule into a cell,comprising contacting the cell with the recombinant AAV viral vector ofany one of embodiments 33 to 43.

52. An AAV viral vector of any one of embodiments 32-43 for use as amedicament.

53. An AAV viral vector of any one of embodiments 32-43 for use in amethod of treatment.

EXAMPLES Example 1. Combinatorial Engineering and Selection of AAVVectors Targeting Ophthalmic Tissues

The method for generating AAV mutants targeting ophthalmic tissues(i.e., the RPE and/or retina) is as follows. The first step involvesidentification of conformational 3D surface-exposed loop on the AAVcapsid surface, for example using cryo-electron microscopy. Selectedresidues within the surface-exposed loop are then subjected tomutagenesis using degenerate primers with each codon substituted bynucleotides NNK and gene fragments combined together by Gibson assemblyand/or multistep PCR. Capsid-encoding genes containing a degeneratelibrary of mutated antigenic motifs are cloned into a wild type AAVgenome to replace the original Cap encoding DNA sequence, yielding aplasmid library. Plasmid libraries are then transfected into 293producer cell lines with an adenoviral helper plasmid to generate AAVcapsid libraries, which can then be subjected to selection. Successfulgeneration of AAV libraries is confirmed via DNA sequencing.

In order to select for new AAV strains that that have desirabletransduction profiles, AAV libraries are subjected to multiple rounds ofinfection in non-human primates. At each stage, tissues of interest areisolated from animal subjects. Cell lysates harvested from the tissuesof interest are sequenced to identify AAV vector isolates successfullytransducing a target tissue of interest. After multiple rounds ofinfection in non-human primates, the isolated sequences from eachmutagenized region are combined in all permutations and combinations.

As a nonlimiting specific example, a surface-exposed loop on an AAVcapsid protein was subjected to mutagenesis as described above toproduce degenerate libraries of AAV vectors. The degenerate librarieswere then subjected to a first round of infection in non-human primates(FIG. 1A, FIG. 2A). Specifically, the libraries were administered to theeye of the primates by intravitreal injection. Retinal pigmentepithelium (RPE) was harvested at day 7 post-infection and sequenced toidentify single AAV isolates (FIG. 1B, FIG. 2B).

The AAVs isolated during the first round of evolution (FIG. 2B) werethen reintroduced into a second non-human primate by intravitrealinjection. Retinal pigment epithelium (RPE) (FIG. 3A) and retina (FIG.4A) were harvested at day 7 post-infection and sequenced to identifysingle AAV isolates.

The AAVs isolated during the second round of evolution in RPE werereintroduced into a third non-human primate, and the AAVs isolatedduring the second round of evolution in retina were reintroduced into afourth non-human primate by intravitreal injection. RPE was harvested atday 7 post-infection from the third primate (FIG. 3B), and retina washarvested at day 7 post-infection from the fourth primate (FIG. 4B), andsequenced to identify single AAV isolates.

A description of the various isolates identified in RPE and/or retina isprovided in Table 7. These isolates specifically target and infect theRPE and/or the retina in vivo, in non-human primates.

TABLE 7 Surface loop substitutions in recombinant AAVsIsolated from RPE and/or retina Sequence Replacing Surface Loop SequenceFull Capsid Sequence KVRDLF (SEQ ID NO: 14) SEQ ID NOs: 32, 41RVLALR (SEQ ID NO: 15) SEQ ID NOs: 33, 42 RVHALR (SEQ ID NO: 16)SEQ ID NOs: 34, 43 RVHSLR (SEQ ID NO: 17) SEQ ID NOs: 35, 44GVGVLP (SEQ ID NO: 18) SEQ ID NOs: 36, 45 FVNALN (SEQ ID NO: 19)SEQ ID NOs: 37, 46 IVRSLN (SEQ ID NO: 20) SEQ ID NOs: 38, 47HVLRLN (SEQ ID NO: 21) SEQ ID NOs: 39, 48 RVLALQ (SEQ ID NO: 22)SEQ ID NOs: 40, 49 RVRGLR (SEQ ID NO: 187) SEQ ID NOs: 195, 203KVRTLR (SEQ ID NO: 188) SEQ ID NOs: 196, 204 MVGNLV (SEQ ID NO: 189)SEQ ID NOs: 197, 205 RVLGLR (SEQ ID NO: 190) SEQ ID NOs: 198, 206KVAGLC (SEQ ID NO: 191) SEQ ID NOs: 199, 207 IVRPLV (SEQ ID NO: 192)SEQ ID NOs: 200, 208 KVRGLA (SEQ ID NO: 193) SEQ ID NOs: 201, 209RVRGLG (SEQ ID NO: 194) SEQ ID NOs: 202, 210

Example 2. Recombinant AAV Vectors Transduce Cells in Culture

To confirm whether various AAV vectors isolated from non-human primateRPE and/or retina in Example 1 are generally infective and able totransduce cells in culture, various AAV vectors packaging a luciferasetransgene were prepared, wherein each AAV vector included a capsidsequence comprising one of the substitutions listed in Table 7. The AAVvectors were contacted with U87 cells (primary glioblastoma cell line)maintained under standard culture conditions. The cells were infected ata multiplicity of infection (MOI) of 10,000 vg/cell. 48 hours later, thecells were lysed, the lysate was contacted with a bioluminescentsubstrate, and RFUs were measured.

As shown in FIG. 5, all AAV vectors tested were able to successfullytransduce U87 cells in culture, resulting in expression of theirpackaged transgene (luciferase) in the cells. This data confirms thatthe recombinant AAV vectors are infective and can be used to deliver atransgene to a cell of interest.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. A recombinant adeno-associated virus (AAV) capsid protein, whereinthe capsid protein comprises a substitution in a surface-exposed loop ofthe AAV capsid protein, wherein the substitution has a sequence of anyone of SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 187, 188, 189,190, 191, 192, 193, or
 194. 2. The recombinant AAV capsid protein ofclaim 1, wherein the substitution has a sequence of any one of SEQ IDNO: 14, 17, 19, or
 22. 3. The recombinant AAV capsid protein of claim 1or 2, wherein the capsid protein comprises an amino acid sequence withat least 80% sequence identity to a capsid protein of any one of AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12,AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV and avian AAV.
 4. Therecombinant AAV capsid protein of claim 1 or 2, wherein the capsidprotein comprises an amino acid sequence that has at least 90% sequenceidentity with any one of SEQ ID NO: 11-12, 23-49, or 195-254.
 5. Therecombinant AAV capsid protein of claim 3, wherein the capsid comprisesan amino acid sequence of any one of SEQ ID NO: 11-12, 23-49, or195-254.
 6. The recombinant AAV capsid protein of claim 5, wherein thecapsid comprises an amino acid sequence of any one of SEQ ID NO. 32, 35,37, or
 40. 7. The recombinant AAV capsid protein of claim 1, wherein theamino acid substitution replaces the amino acids in the regioncorresponding to amino acid positions 591-596 of SEQ ID NO. 11 or aminoacids 590-595 of SEQ ID NO.
 12. 8. A recombinant AAV capsid protein,wherein the capsid protein comprises a substitution comprising asequence of six amino acids (X¹-X²-X³-X⁴-X⁵-X⁶) that does not occur inthe native AAV capsid protein, wherein X² is V and X⁵ is L (SEQ ID NO:186).
 9. The recombinant AAV capsid protein of claim 8, wherein thesubstitution replaces the amino acids in the region corresponding toamino acid positions 591-596 of SEQ ID NO. 11 or amino acids 590-595 ofSEQ ID NO.
 12. 10. The recombinant AAV capsid protein of claim 8 or 9,wherein the substitution is in a surface-exposed loop of the AAV capsidprotein.
 11. The recombinant AAV capsid protein of any one of claims8-10, wherein X¹ is not T, X³ is not D, X⁴ is not R, and X⁶ is not T.12. The recombinant AAV capsid protein of any one of claims 8-11,wherein X¹ is K, G, F, I, H, or R.
 13. The recombinant AAV capsidprotein of any one of claims 8-11, wherein X³ is R, L, H, G, or N. 14.The recombinant AAV capsid protein of any one of claims 8-11, wherein X⁴is D, A, S, or V, or R.
 15. The recombinant AAV capsid protein of anyone of claims 8-11, wherein X⁶ is F, R, P, N, or Q.
 16. The recombinantAAV capsid protein of any one of claims 8-11, wherein X¹ is K, X³ is R,X⁴ is D, and X⁶ is F.
 17. The recombinant AAV capsid protein of any oneof claims 8-11, wherein X¹ is R, X³ is L, X⁴ is A, and X⁶ is R.
 18. Therecombinant AAV capsid protein of any one of claims 8-11, wherein X¹ isR, X³ is H, X⁴ is A, and X⁶ is R.
 19. The recombinant AAV capsid proteinof any one of claims 8-11, wherein X¹ is R, X³ is H, X⁴ is S, and X⁶ isR.
 20. The recombinant AAV capsid protein of any one of claims 8-11,wherein X¹ is G, X³ is G, X⁴ is V, and X⁶ is P.
 21. The recombinant AAVcapsid protein of any one of claims 8-11, wherein X¹ is F, X³ is N, X⁴is A, and X⁶ is N.
 22. The recombinant AAV capsid protein of any one ofclaims 8-11, wherein X¹ is I, X³ is R, X⁴ is S, and X⁶ is N.
 23. Therecombinant AAV capsid protein of any one of claims 8-11, wherein X¹ isH, X³ is L, X⁴ is R, and X⁶ is N.
 24. The recombinant AAV capsid proteinof any one of claims 8-11, wherein X¹ is R, X³ is L, X⁴ is A, and X⁶ isQ.
 25. The recombinant AAV capsid protein of any one of claims 8-24wherein the AAV capsid protein is of an AAV serotype selected from AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12,AAVrh.8, AAVrh.10, AAVrh32.33, AAVrh74, bovine AAV and avian AAV.
 26. Arecombinant AAV capsid protein comprising the amino acid sequence of anyone of SEQ ID NO: 11-12, 23-49, or 195-254.
 27. The recombinant AAVcapsid protein of claim 26 comprising the amino acid sequence of SEQ IDNO: 32, 35, 37, or
 40. 28. A nucleotide sequence encoding an AAV capsidprotein of any one of claims 1-27.
 29. The nucleotide sequence of claim28, wherein the nucleotide sequence is a DNA sequence.
 30. Thenucleotide sequence of claim 28, wherein the nucleotide sequence is anRNA sequence.
 31. An expression vector comprising the nucleotidesequence of any one of claims 28-30.
 32. A cell comprising thenucleotide sequence of any one of claims 28 to 30 or the expressionvector of claim
 31. 33. A recombinant AAV viral vector comprising thecapsid protein of any one of claims 1 to
 27. 34. The recombinant AAVviral vector of claim 33, wherein the AAV viral vector has an enhancedtransduction profile with respect to a target tissue, as compared to anAAV viral vector comprising a capsid protein that does not comprise thesubstitution.
 35. The recombinant AAV viral vector of claim 34, whereinthe target tissue is an ophthalmic tissue.
 36. The recombinant AAV viralvector of any one of claims 33-35, further comprising a cargo nucleicacid encapsidated by the capsid protein.
 37. The recombinant AAV viralvector of claim 36, wherein the cargo nucleic acid encodes a therapeuticprotein or RNA.
 38. The recombinant AAV viral vector of claim 36 or 37,wherein the cargo nucleic acid encodes one or more of the followingproteins or an antibody that binds thereto: cystic fibrosistransmembrane regulator protein (CFTR), dystrophin, myostatinpropeptide, follistatin, activin type 11 soluble receptor, IGF-I, IkappaB dominant mutant, sarcospan, utrophin, mini-utrophin, Factor VIII,Factor IX, Factor X, erythropoietin, angiostatin, endostatin, catalase,tyrosine hydroxylase, superoxide dismutase, leptin, the LDL receptor,lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin.spectrin, alpha-1-antitrypsin, adenosine deaminase, hypoxanthine guaninephosphoribosyl transferase, β-glucocerebrosidase, sphingomyelinase,lysosomal hexosaminidase A, branched-chain keto acid dehydrogenase,RPE65 protein, alpha-interferon, beta-interferon, gamma-interferon,interleukin-2, interleukin-4, granulocyte-macrophage colony stimulatingfactor, lymphotoxin, a peptide growth factors, a neurotrophic factors,somatotropin, insulin, insulin-like growth factors 1 or 2, plateletderived growth factor, epidermal growth factor, fibroblast growthfactor, nerve growth factor, neurotrophic factor -3 or -4, brain-derivedneurotrophic factor, RANKL, VEGF, glial derived growth factor,transforming growth factor-alpha or beta, lysosomal acidalpha-glucosidase, alpha-galactosidase A, tumor necrosis growth factorsoluble receptor, S100A1, parvalbumin, adenylyl cyclase type 6, SERCA2A,Inhibitor 1 of PP 1 or fragments thereof, truncated constitutivelyactive bARKct, IRAP, anti-myostatin protein, aspartoacylase,trastuzumab, galanin, Neuropeptide Y, Vasohibin 2, thymidine kinase,cytosine deaminase, diphtheria toxin, tumor necrosis factor, p53, Rb,Wt-1, TRAIL, RS1, opsin, TKR-beta, C3, CFH, and/or FAS-ligand.
 39. Therecombinant AAV viral vector of claim 36 or 37, wherein the cargonucleic acid encodes a gene-editing molecule.
 40. The recombinant AAVviral vector of claim 39, wherein the gene-editing molecule is anuclease.
 41. The recombinant AAV viral vector of claim 39 or 40,wherein the gene-editing molecule is a Cas9 nuclease.
 42. Therecombinant AAV viral vector of claim 39 or 40, wherein the gene-editingmolecule is a Cpf1 nuclease.
 43. The recombinant AAV viral vector ofclaim 39, wherein the gene-editing molecule is a guide RNA.
 44. Apharmaceutical composition comprising the recombinant AAV viral vectorof any one of claims 33 to
 43. 45. The pharmaceutical composition ofclaim 44, wherein the composition further comprises a pharmaceuticallyacceptable carrier.
 46. A method of treating a patient in need thereofcomprising administering to the patient a therapeutically effectiveamount of a recombinant AAV viral vector of any one of claims 33 to 43.47. The method of claim 46, wherein the patient has a disease ordisorder of the eye.
 48. The method of claim 47, wherein the disease ordisorder of the eye is retinitis pigmentosa, macular degeneration, opticneuritis, Leber's congenital amaurosis, Leber's hereditary opticneuropathy, achromatopsia, X-linked retinoschisis, optic neuritis,choroideremia, optic atrophy, retinal cone dystrophy, retinopathy,retinoblastoma, glaucoma, Bardet-Biedl syndrome, Usher syndrome,aniridia, Friedreich's ataxia, vitelliform macular dystrophy,retinoblastoma, Stargardt disease, Charcot-Marie-Tooth disease, Fuch'sdystrophy, propionic acidemia, or color blindness.
 49. The method of anyone of claims 46-48, wherein the patient is a mammal.
 50. The method ofclaim 49, wherein the patient is a human.
 51. A method of introducing anucleic acid molecule into a cell, comprising contacting the cell withthe recombinant AAV viral vector of any one of claims 33-43.
 52. An AAVviral vector of any one of claims 32-43 for use as a medicament.
 53. AnAAV viral vector of any one of claims 32-43 for use in a method oftreatment.